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Additional Information
Safe approaches for camptothecin delivery: structural analogues and
nanomedicines

Pablo Botella * and Eva Rivero-Buceta

Instituto de Tecnología Química, Universitat Politècnica de València-Consejo Superior de

Investigaciones Científicas, Avenida de los Naranjos s/n, 46022 Valencia, Spain

* To whom correspondence should be addressed. E-mail: [email protected]. Fax: +34 96 3879444.

Abstract

Twenty-(S)-Camptothecin is a strongly cytotoxic molecule with excellent antitumor

activity over a wide spectrum of human cancers. However, the direct formulation is

limited by its poor water solubility, low plasmatic stability and severe toxicity, which

currently limits its clinical use. As a consequence, two strategies have been developed

in order to achieve safe and efficient delivery of camptothecin to target cells: structural

analogues and nanomedicines. In this review we summarize recent advances in the

design, synthesis and development of camptothecin molecular derivatives and

supramolecular vehicles, following a systematic classification according to structure-

activity reationships (structural analogues) or chemical nature (nanomedicines). A series

of organic, inorganic and hybrid materials are presented as nanoplatforms to overcome

camptothecin restrictions in administration, biodistribution, pharmacokinetics and

toxicity. Nanocarriers which respond to a variety of stimuli endogenously (e.g., pH, redox

potential, enzyme activity) or exogenously (e.g., magnetic field, light, temperature,

ultrasound) seem the best positioned therapeutic materials for optimal spatial and

temporal control over drug release. The main goal of this review is to be used as a source

of relevant literature for othersinterested in the field of camptothecin-based

therapeutics.To this end, final remarks on the most important formulations currently

under clinical trial are provided.

Keywords: camptothecin analogues; camptothecin nanomedicines; structure-activity

relationship; stimuli-responsive; clinical trials

Contents

1. Introduction

2. Camptothecin analogues

2.1. Structure-activity relationship

2
2.2. Camptothecin structural derivatives at the clinical stage

3. Nanoparticles as camptothecin delivery systems

3.1. Organic nanocarriers

3.1.1. Micelles

3.1.2. Liposomes

3.1.3. Dendrimers

3.1.4. Organic polymers and biopolymers

3.2. Inorganic nanocarriers

3.2.1. Metal nanoparticles

3.2.2. Carbon nanotubes

3.3. Hybrid nanocarriers

3.3.1. Organic-inorganic nanoparticles

3.3.2. Core-shell systems

3.3.3. Metal-organic framework nanoparticles

4. Stimuli-responsive systems

4.1. pH-sensitive systems

4.2. Redox-sensitive systems

4.3. Enzyme-sensitive systems

4.4. Light-sensitive systems

4.5. Magnetic-sensitive systems

4.6. Other stimuli-responsive systems for camptothecin delivery

5. Clinical testing

3
6. Conclusions and future directions

Acknowledgements

References

1. Introduction

Camptothecin (CPT) is a water insoluble, natural pentacyclic alkaloid isolated from the

oriental tree Camptotheca accuminata by Wall et al. in 1966 [1]. CPT is well-known for

its antitumor activity against a wide spectrum of human cancers [2-8]. In addition, a

variety of biological activities, such as pesticidal [9-10], antipsoriasis [11-12], antiparasitic

[13], antifungal [14], antimicrobial [15] and antiviral [16-17] has been described for this

compound. Certainly, several researchers have reported that CPT inhibits a cellular

enzyme DNA topoisomerase I and induces apoptosis in various cancer cells. [18-19]

CPT is a planar pentacyclic quinoline that includes 3 rings of pyrrolo- (3,4-β)- quinoline

part (A, B, and C rings), an unsaturated pyridone moiety in ring D and a α-hydroxy lactone

ring (E ring), containing one chiral center with (S)-configuration (Fig. 1) [20-21]. It is worth

noting that the isomer 20(R) hydroxyl has little activity while the isomer 20(S) is between

10 and 100 fold more active [22]. Also, it is known that E-ring exists in equilibrium

between the lactone form (closed ring, not water insoluble) and the carboxylate form

(open ring, water soluble). In acidic pH the lactone predominates which, according to

different clinical trials and structure-activities studies, is the only therapeutic molecule.

O O
5 OH
7
9 N 22 N
C
B D OH O
10
A E O
N 20
N
14 H
11 S´ O
12 O
OH OH

Lactone (close ring) Carboxylate (open ring)

Fig. 1. CPT equilibrium between lactone (active form) and carboxylate (inactive form)

4
However, at physiological pH, the lactone ring is converted into the carboxylate, much

less active, which predominates at neutral and alkaline pH [23-24].

Unfortunately, CPT presents some major limitations with regards to therapeutic

application, like poor water solubility and the rapid lactone ring hydrolysis at physiological

pH, which gives rise to the inactive carboxylate form [25]. In addition, CPT is extremely

insoluble in organic compounds except for dimethyl sulfoxide, in which it shows moderate

solubility [26]. Due to CPT insolubility in biocompatible solvents, it is very difficult to apply

conventional drug administration routes, including oral, intravenous or intramuscular

injection, to distribute this compound throughout the body [27]. Furthermore, there are

other negatives aspects that limit the use of CPT in clinical trials: pronounced loss of

activity due to lactone-ring hydrolysis, reversibility of drug-target interaction and severe

toxicity, including hemorrhagic cystitis and myelotoxicity [28]. All these drawbacks have

precluded its clinical application, making necessary to develop alternative compounds

and structures for CPT use in humans.

In an effort to improve CPT solubility and pharmacokinetics structural derivatives as

topotecan and irinotecan have been developed [29-30] (see Table 1). Unfortunately, as

it will be reported later on, CPT quinoline structure modification usually brings out a

dramatic drop of cytotoxic activity, which results one order or even lower than pristine

drug, strongly limiting the therapeutic use.

To overcome these issues two strategies have been pointed out for safety and efficient

CPT delivery to target cells: i) structural analogues, in which the CPT molecule is

chemically modified for increased solubility and stability in biological fluids; and ii)

nanomedicines, wherein CPT is incorporated by physico-chemical methods to

nanoparticles which act as stable carriers for drug delivery. In this review, we summarize

the most advanced and recent achievements in the design, synthesis and development

of CPT analogues and nanomedicines, including the most relevant formulations currently

under clinical use.

5
2. Campthotecin analogues

Since the discovery of CPT and the associated therapeutic limitations, huge scientific

efforts are being made to improve the pharmacokinetics, drug resistance, clinical

efficacy, and toxicity profiles of the original molecule, by introducing organic ligands [31-

36].

2.1. Structure-activity relationship

In literature, numerous structure-activity relationship (SAR) studies have focused in

different modifications of A, B, C, D and E rings of CPT to obtain more active analogs

(Table 1 and Fig. 2). It was reported that the complete pentacyclic ring structure was

essential for its activity and the planar structure of this system was suggested to be one

of the most influential structural element. In addition, the hydroxylactone ring is the most

critical region for the activity [35,37-38].

Most derivatives of CPT have been obtained through modifications of the quinolone A-B

ring. However, to date, only two of these CPT analogues have been approved for clinical

use. Modifications may include substitution at 7, 9, 10 or 11 positions [2, 39-43]. In

general, substitution at C12 is unfavorable to biological activity. This loss in the activity

can be due to steric and electronic disturbances at the quinoline nitrogen, which might

interfere interaction with DNA [44]. Monosubstitution at C9, C10 or C11 by amine or

hydroxyl group make it possible to obtain compounds with more antitumoral activity,

whereas substitution of positions 9 and 10 by halides or other electron-rich groups and

substitution of position 11 with fluorine or cyano groups increase the DNA topo I inhibition

ability [44-45]. Moreover, modifications at C10 and C11 positions are adverse for

antitumor activity with the exception of the 10,11-methyl or ethylenedioxy, substitutions

favorable to DNA topo I inhibition [46-47]. Conversely, other ring combinations in

6
positions 7 and 9 do not positively affect DNA topo I activity, whereas substitutions at C7

and C10 showed powerful cytotoxicity [48-49].

Table 1. CPT analogues. Most important structural modifications in A, B, C, D, E rings.

Prodrug Substituent position IC50a MTDb Clinical Ref.
C11 C10 C9 C7 (µM) (mg/m /d)2

Irinotecanc O
H N N H CH2CH3 1.14 290-320 Approved [30,60]
(CPT-11) O

SN38 H OH H CH2CH3 0.09 n/a - [61-63]

Topotecanc H OH N H 0.1 1.5 Approved [30,36]

Rubitecan H H NO2 H 0.085 1.5 Phase III [36,62]

(9-NC)
Belotecan
H H H N 0.094 0.5 Phase II [68-69]
(CKD-602) H

Exatecan NH
F CH3 __ 0.008 0.3-0.5d Phase III [34,70]
(DX-8951f)
Lurtotecan O O
__ H N NCH3 0.006 1.2 Phase II [34,71]
(GG-211)

CPTc H H H H 0.046 - [36]

a IC : drug concentration needed to inhibit 50% of the cell growth compared to growth of the untreated
50
control cells. All compounds were tested in a variety of human tumor cell lines.
b
MTD: Maximun tolerated dose
c Solubility in water at 25 ºC: Irinotecan (HCl)=25 mM; Topotecan (HCl)=100 mM; CPT= 2.9 mM.
d
MTD: 0.3 mg/m2/day for heavily pretreated patients and 0.5 mg/m2/day for minimally pretreated patients.

N
N
O
O N
N -
O +O
Irinotecan O N

OH O O
N
HO N
O
N O
N N
Rubitecan
NH O OH O
O
SN-38 N
O OH O
N
N C9 O
N C7 + C10
O OH O
C7 10-(4-pyridyl)-CPT
7 5 C10
OH O 9 O
Belotecan
10 C N
A B 16 22
D N
C5+C9 N
11 E O C9+C10
OH 12 1 14 HO
20 O
O N
N C10 + C11 R1 O N
N C11 O
O Topotecan
O O OH O
OH O N
5-ethyl-9-hydroxy-CPT O N O
O N
R1-4 N
10,11-methylenedioxy-CPT
OH O O

R1=OH OH O
O
O
N R2= F
O N R3=CN
O R4=NH2
10,11-ethylenedioxy-CPT
OH O

Fig. 2. Scheme of different CPT structural modifications. Where possible, the structure
of the resulting analogue is depicted.

7
Also, a series of 7-substituted CPTs were developed to overcome the instability of

lactone ring. All of them showed significantly improved cytotoxic/antitumor potency and

stability [50]. On the other hand, few studies have focused on modifications of C and D

rings. In general, modifications in position 5, 14 and 17 of these rings reduce cytotoxicity

as CPT loses its planarity. Substitution of alkoxy or several other groups at position 5 are

reasonably well tolerated when the substituents at position 9 are nitro or hydroxyl groups

[51]. Besides, reduction of carbonyl at C17 leads to inactive compounds [34-35,52-54].

At this point, it must be emphasized that E ring and stereochemistry at 20 position are

crucial for CPT activity. One of the major drawbacks observed in ring E modification is

that, due to the presence of α-OH group, under physiological conditions the lactone ring

is cleavaged to inactive carboxylate form. Here, with the aim of increasing lactone ring

stability, several derivatives have been reported but with lower cytotoxicity. Also, it is

noteworthy that α-hydroxylactone replacement by β-hydroxylactone improves lactone

cytotoxicity and stability [35,38].

More recent studies have been focused on different conjugates (ester, amide, carbonate,

etc.) at C20 position aimed at new CPT structures. As it will be detailed later on (section

3.1), CPT can be covalently linked by 20-hydroxyl group to numerous organic moieties,

including glycerides, poly(amidoamine) and triazine derivatives, polyethylene glycol,

poly(N-(2-hydroxypropyl) methacrylamide), polyvinylpyrrolidone, polyethyleneimine,

poly(L-glutamic acid), β-cyclodextrins, hyaluronic acid, chitosan and polypeptides.

Actually, these systems improve CPT delivery and bioavailability, and may also introduce

alternative administration routes to parenteral injection [32,55-58].

2.2. Camptothecin structural derivatives at the clinical stage

Although a variety of CPT derivatives have been developed, only a short list has been

investigated in clinical trials [59]. Herein, we try to mention the most representative CPT-

8
based molecules at the clinical stage. Such illustrative examples are presented in Table

1.

In particular, CPT-11 (also known as irinotecan) was approved by the Food and Drug

Administration (FDA) and is used to treat colorectal and other cancers. In vivo, CPT-11

is converted into the active metabolite SN38 by the hepatic carboxylesterase enzyme.

This metabolite allows to overcome the problems of irinotecan, including intestinal

toxicity and neutropenia [60]. Furthermore, SN-38 is one of the most active analogues

and shows higher cytotoxic activity than irinotecan [61-62]. However, the principle issue

of SN38 is its poor water solubility [63].

Topotecan was the first topoisomerase I inhibitor approved by the FDA and is used to

treat ovarian and lung cancers. This compound has shown activity against a broad

spectrum of cancers cell lines. A report about Phase II testing [64], mentions that the

efficacy of topotecan as second treatment in ovarian cancer was demonstrated among

92 patients. Although this compound was tolerated but different side effects such as

alopecia, nausea and vomiting appeared.

Another CPT derivative is rubitecan (9-nitrocamptothecin, 9-NC), a water insoluble

analogue currently in Phase III trial. This compound can be metabolically converted in

vivo to 9-aminocamptothecin (9-AC), which showed anticancer activity in Swiss

immunodeficient (nude) mice of the NIH-1 high fertility strain inoculated with different

breast carcinoma cell lines (MDA-MD-134 and MDA-MB-231), at a concentration

several-fold lower than CPT [65]. Also, rubitecan, showed similar activity than CPT

against a broad spectrum of tumor types in human tumor xenograft models [66], and in

a Phase II trial over 58 advanced pancreatic carcinoma patients, it achieved >50% tumor

shrinkage in 7 cases (16%), being well tolerated by most of patients (only 3 patients

discontinued treatment due to toxicity) [67].

Conversely, belotecan (CKD-602) is another derivative of CPT in Phase II that solves

the problem of water solubility. This compound showed more activity than topotecan

9
against a broad spectrum of cancers cell lines and acceptable toxicity [68]. Recently,

different clinical studies of belotecan in combination with other chemotherapy drugs such

as cisplatin have been investigated [69]. This pharmacological combination

demonstrated a promising efficacy in patients with advanced stage small cell lung

cancer, although the hematologic toxicity of this regimen requires substantial attention.

Exatecan (DX-8951f) is an intrinsically active compound also in Phase II. This CPT

analogue showed higher efficacy against human tumor xenografts (colon, lung, breast,

renal) than topotecan and irinotecan. [70]. In an experiment that included over 31

patients with different solid malignancies, the administration of exatecan as a protracted

21-day continuous i.v. infusion showed limited systemic toxicity (with a significant

reduction of myelosuppresive effects, neutropenia and thrombocytopenia) and efficient

anticancer activity.

Finally, lurtotecan (GG-211) is also a water soluble CPT analogue currently in Phase II.

This compound showed significant cytotoxicity against HT-29 and SW-48 human colon

cancer cells, PC-3 human prostate cancer cell line, MX-1 human breast cancer cells,

H460 human lung cancer cell line, SKOV3 human ovarian cancer cells and KB human

epidermoid carcinoma cell line [71].

3. Nanoparticles as drug delivery systems for camptothecin

Many types of nanovehicles have been developed for CPT delivery, and these drug

delivery systems (DDSs) may be classified in several ways, according to their chemical

nature, structure, composition, morphology, etc. Herein, we present a systematic

classification of CPT nanocarriers according to their chemical nature in organic, inorganic

and hybrid materials, presenting different properties and behaviors [72-75]. These novel

nanoplatforms have been designed to solve most of the antitumor drug limitations,

improving CPT pharmacokinetics, which is affected by poor water solubility, rapid plasma

10
clearance, high systemic toxicity and poor selectivity towards cancer cells [36]. A variety

of DDSs have been developed and allow CPT incorporation. In addition, these DDSs

can conjugate the drug in different ways, as adsorption, encapsulation or covalent

bonding [76], working as inert supports to transport the drug to tumor [77]. In this sense,

some major advantages of these DDSs over free CPT are: improved solubility, lactone

ring stability, half-life extension, biocompatibility and control drug release rates, which

allow significant advances at the clinical stage [78-79].

3.1. Organic nanocarriers

The use of organic pharmaceutical nanocarriers to enhance the in vivo efficacy of many

drugs has been well established in the last two decades [80]. To prepare such smart

systems, therapeutic moieties must be simultaneously encapsulated or assembled on

the surface [81]. Organic carriers are carbon-based (with the exception of carbon

nanotubes that are considered inorganic) and are generally characterized by their high

biocompatibility, good solubility or colloidal stability in physiological fluids and quick

biodegradability, resulting in excellent pharmacokinetic profiles of the delivered drugs.

However, their usual limitations include restricted functionalization (with hinders not only

drug loading, but also the incorporation of targeting molecules and cromophores), low

stability (especially under enzymatic activity), and the lack of specific physicochemical

properties, as optical activity or magnetism, which are useful in diagnostics [82]. In these

materials, drugs are often trapped or bound within the matrix.

Although different classifications may be used, we have organized the various organic

nanocontainers according to their chemical nature, resulting in five key groups: micelles,

liposomes, dendrimers and polymers and biopolymers.

11
3.1.1. Micelles

Polymeric micelles are expected to increase the accumulation of drugs in tumor tissues

by taking advantage of the enhanced permeability retention (EPR) effect [83]. Actually,

micelle systems can also incorporate various kinds of drugs into their inner core with

relatively high stability by chemical conjugation or physical entrapment [84-85]. Also, the

size of micelles can be controlled within the diameter range of 20-100 nm. Therefore, it

is expected that the incidence of drug-induced side effects may be decreased due to

reduced drug distribution in normal tissues [86].

At this point, polymeric micelles composed of various poly(ethylene glycol)-

poly(aspartate ester) block copolymers have been physically loaded with CPT, to

improve drug aqueous solubility and stability in biological fluids [87-89]. Here, it was

observed that the stability of CPT-loaded micelles depended on the amount of benzyl

esters and poly(ethylene glycol) (PEG) length in the polymers. After intravenous

administration of CPT-loaded micelles to male ddY mice via lateral veins, it was

concluded that sample using PEG 5000, 27 aspartic acid (Asp) units and 57-75% benzyl

esterification of Asp residue was the most stable formulation, showing an extended

circulation period in blood stream [82]. However, it is not clear if CPT is safely packaged

in these micelles, which is crucial to avoid premature release before reaching tumor cells.

Another nanomicelle system was developed by using α,β-poly[(N-carboxybutil)-L-

aspartamide] (PBAsp) with different CPT loading (PBAsp-CPT), through esterification

between PBAsp carboxyl groups and CPT 20-hydroxyl [90-91] (Fig. 3). Subsequently,

spherical nanomicelles were produced in aqueous medium. In this case, CPT was

sequestered toward the core whereas PBAsp backbone formed the hydrophilic shell.

Unfortunately, PBAsp-CPT solubility decreased quickly as the amount of CPT increased.

Anyway, in vitro cytotoxicity assays of PBAsp-CPT (2,1% CPT) against L929 mouse

muscular cell line showed less toxicity than free CPT (5-10% activity drop depending of

CPT concentration) due to its linear sustained drug release profile.

12
 O
O

NH
NH
O O
β
HN α HN
O

OH

O
O
O

O
N
N
O

Fig. 3. Schematic illustration of the conjugate made of α,β-poly[(N-carboxybutil)-L-

aspartamide] and CPT. (Reprinted from ref. 90, Copyright © 2010, with permission from
Elsevier)

In addition, CPT was also encapsulated in different quantities in 45 nm diameter micelles

of poly(ethylene glycol)-poly(L-lysine) block copolymer and 3-3’-dithiodipropionic acid.

Then, it was studied CPT release in response to cytosol reductive conditions was studied

by exploiting the permeabilization of endosomes upon light irradiation (using a halogen

lamp with a 400-700 nm band-pass filter) induced by photosensitizer agents, such as

Photofrin. Experiments in BALB/c nu/nu mice inoculated subcutaneously with AY27

urothelial cancer cells confirmed that after endosomal permeabilization CPT loaded

micelles escaped (from endocytic vesicles) into the cytosol, promoting drug release of

more than 70% from micelles in the irradiated tissue (fluence rate to induce the

photochemical internalization effect, 100 mW/cm2; time, 1000 s) and better antitumor

activity than the free drug [92].

3.1.2. Liposomes

Liposomes are a well-studied biocompatible carrier playing a key role in nanotechnology-

based drug delivery [93]. Liposomes protect the encapsulated drugs form structural

transformation or chemical degradation by isolating them from the surrounding

environment. However, formulating CPT liposomes is challenging due to different

technical issues, as the drug shows poor solubility in nearly all pharmaceutically

13
acceptable solvents, as well as it associates with simple phospholipid bilayers. Actually,

the outcome in most of these approaches is hampered by incomplete separation of free

and liposomal CPT, which affects to biological activity and biodistribution. In that sense,

a study over several CPT liposome formulations of different nature (cationic, anionic and

neutral phospholipids) prepared by drug encapsulation, reported cationic compositions

as the best option to increase CPT water solubility [94].

In one of the first attempts to deliver CPT within liposomes, the lipid-complexed

formulation (particle size range 20-200 nm) showed significant advantages with regards

to the naked drug for intravenous administration in clinically relevant lipid-drug ratios

(12.5:1 w/w) [95]. CPT-loaded liposomes (LC-CPT) had an in vitro antitumor activity

similar to that of free CPT and displayed similar cytotoxicity against multidrug resistance

(MDR-1) negative human cervix carcinoma KB-V1 cells and MDR-1 positive human

cervix carcinoma KB-3-1 cell line. However, the biodistribution of CPT in ICR Swiss mice

was severely affected by lipid complexation, as CPT achieved the greatest concentration

in pulmonary parenchyma, while liposomes accumulated mostly at gastrointestinal tract,

probably due to the rapid processing of LC-CPT in liver and elimination via the gut.

Furthermore, LC-CPT showed higher antitumor activity than free CPT in in vivo testing

over B6D2F1 mice bearing intraperitoneal tumors of L1210 mouse lymphocytic leukemia

cells or P388 mouse leukemia cell line.

CPT-loaded liposomes with average particle size of about 150 nm were synthesized by

a lipid-film hydration method [96]. In a first step, liposomes were formulated by addition

of an artificial lipid with a phenyl group (3,5-bis(dodecyloxy)benzoic acid) to polyethylene

glycol-modified liposomes and, subsequently, liposome surface was coated with human

serum albumin. Here, CPT was entrapped with about 80% efficiency due to the

interaction with phenyl group-containing lipids. After intravenous injection, these

protected liposomes showed much higher therapeutic activity than CPT in CDF1 female

mice bearing mouse colon adenocarcinoma 26 tumors (tumor volume in CPT-treated

14
specimens increased up to 5-fold that of liposome-treated mice), with high accumulation

in malign tissue (9.6-fold more efficiently than CPT solution).

3.1.3 Dendrimers

Dendrimers have received much attention in drug delivery, as they are highly branched,

multivalent and tunable polymers for different architecture, size, shape and surface

properties [97]. Dendrimers are usually classified by generation (denominated G), which

refers to the number of repeated branching cycles that are performed during its

synthesis. Due to their positive charge, cationic dendrimers as poly(amidoamine)

(PAMAM) and triazine derivatives conjugate easily with the phenolic 20-hydroxyl group

of CPT, resulting in stable nanomedicines. At this point, it has been proved that CPT

solubility increases with PAMAM generation [98]. Here, PAMAM dendrimers of four (G4),

five (G5) and six (G6) generation were prepared and their solubility in aqueous medium

and the interaction with CPT (carboxylate form or lactone form) were evaluated. The

study showed that G6 PAMAN enhanced CPT water solubility in comparison with G4

PAMAN. This can be partly explained by the fact that the number of primary and tertiary

amines in G6 dendrimer increases and could entrap more hydrophobic molecules inside.

However, the cationic nature of dendrimers represents a serious toxicity issue, as it

promotes a strong reaction with serum proteins, hemolysis and hepatic toxicity.

Furthermore, their chronic toxicity is still to be determined [99].

A relevant PAMAM second generation (G2) model was synthesized with four molecules

of CPT per unit and a trigger that allows activation with penicillin-G-amidase (PGA). In

this system, the cleavage of the phenylacetamide group by PGA triggered the

disassembly of dendron through a known self-immolative reaction sequence (Fig. 4)

[100]. This conjugate demonstrated outstanding improvement of CPT cytotoxic activity,

as cell-growth inhibition assays indicated that half maximal inhibitory concentration (IC50)

value for the dendrimer was between 100- and 1000-fold lower than free CPT against

15
MOLT-3 lymphoblastic leukemia, JURKAT human leukemia T and HEK-293 human

kidney embryonic cell line.

OH

N O

n
N
O
O

O O
O NH
O
N O

O NH
N
O N N

HN
O N O N
O
O N O O
H H N
N O O N
O N O
N O
O N O O O O
O
O
O N
N O
N N O N
O O N
O H O
OO
O
O O
NH N N
O NH O N O
N H O OH
n
N O

O
O N
O N
O

Fig. 4. Molecular structure of second-generation PAMAM with a trigger designed for

activation by penicillin-G-amidase. The figure shows CPT in blue, penicillin-G-amidase
in red, dendritic structure in black and poly(ethylene glycol)-azide units in purple.
(Reprinted with permission from ref. 100. Copyright © 2006 American Chemical Society)

In one important contribution, triazine dendrimer CPT derivatives were synthesized and

their cytotoxic activity evaluated against MCF-7 human breast carcinoma cells and HT-

29 colon carcinoma cell lines [101]. For this sake, CPT was conjugated with isonipecotic

acid through an ester bond, and the obtained secondary amine was highly reactive and

combined with the triazine, allowing to obtain the desired dendrimers, which showed

almost similar cytotoxicity to free CPT.

Click chemistry has been widely used to improve coupling reaction efficacy in the

synthesis of CPT-dendrimer conjugates [102-103]. In a relevant study, CPT was

esterified with a spacer 1-azido-3,6,9,12,15-pentaoxaoctadecan-18-oic acid (APO). On

the other hand, propargylamine (PPA) and methoxypoly(ethylene glycol) amine were

conjugated to PAMAM dendrimer G4.5. Then, CPT-APO was coupled to the modified

16
dendrimer (G4.5-PPA) via click chemistry. Nearly 100% of the CPT molecules were

covalently conjugated to the dendrimer and the drug was released by ester bond

hydrolysis. This new dendrimer-CPT was more cytotoxic than free CPT (IC50 value

increase of 185-fold in U1242 human glioma cells).

3.1.4. Organic polymers and biopolymers

In the last years, a myriad of polymer therapeutics, including polymeric drugs, polymer-

protein conjugates, polyplexes, polymeric micelles and polymer-drug conjugates [104-

105], have received special attention as drug delivery systems and have been

established as first-generation nanomedicines. Most of these polymers present

advantages such as absence of toxicity, biocompatibility, biodegradability and small

particle size, allowing them to circulate in the bloodstream for long periods of time [106-

107]. Conversely, the main issues of these materials are their fast biological degradation,

which may cause leaching of CPT important quantities of CPT into the blood stream and

interstitial fluids, and their variable loading capability due to limited functionalization.

Nanoparticles of synthetic polymers, including polyethylene glycol, poly(N-(2-

hydroxypropyl) methacrylamide), polyvinylpyrrolidone, polyethyleneimine, poly(L-

glutamic acid) and β-cyclodextrins, and natural polymers such as hyaluronic acid,

chitosan and polypeptides, are the most commonly reported for CPT delivery [108-109].

In addition, the presence of a covalent chemical bond between a water-soluble polymeric

carrier and the bioactive molecule confers enhanced solubility and stability, allowing to

improve the pharmacokinetic profile of the bioactive molecule [36, 110-112].

CPT can be encapsulated in hydrophobic polymers or amphiphilic copolymers for safety

delivery. At this point, one interesting approach that allows exceptionally high drug

loading is to polymerize the drug itself. In one representative example, a dimeric CPT

derivative, CPT-SS-CPT, bearing a trigger responsive domain was used as the core-

constructing unit of the nanoparticle [113]. Here, CPT molecules are stably conjugated

via carbonate linkages that are subject to triggered bond cleavage and subsequent drug

17
release by a reducing reagent (Fig. 5). Upon triggering, cleavage of the disulfide bond in

CPT-SS-CPT would result in decomposition of the drug dimer, releasing CPT in its

authentic form. Unfortunately, CPT-SS-CPT has much lower water solubility (less than

10 ng/mL) than CPT (3 μg/mL), presumably due to stronger intermolecular interactions

between molecules. However, when CPT-SS-CPT was encapsulated in methoxy

poly(ethylene glycol)-poly(lactide) (mPEG-PLA, Mw 3500-5000) empty micelles, drug-

loaded nanoparticles stable in phosphate buffer saline (PBS) and human serum for at

least 3 days were formed, with particle diameter of about 180 nm. The anticancer effect

of these CPT-SS-CPT/mPEG-PLA nanoparticles was then evaluated in vitro by MTT

assay over HeLa human cervix carcinoma cell line. The IC50 of CPT-SS-CPT/mPEG-

PLA was 114 nM, compared to 17 nM for free CPT, but the main advantages of this

nanoconjugate are the lack of systemic toxicity, as CPT release only takes place through

disulfide bond reduction by intracellular thiols, and the stabilization of the lactone ring.

O O N3
S
S O -
O S
O O O
O O O O O
O O O NH O NH O
O O O
O O O O
O O O O O
O N O N
N N O
N N
N O
N S

CPT-SS-CPT

O O O
O O H 2N O
O
O O O O
O N
N
N
N

O
N
O N O + CO2
O
OH CO2 O O O
O O NH2 O OH
O NH2 O O
O O O
N HO O O N
N
N H2O
N
N

Fig. 5. Chemical structure of dimeric CPT derivative, CPT-SS-CPT and release

mechanism of thiol-triggered drug release. (Reprinted partially with permission from ref.
113. Copyright © 2015 American Chemical Society)

18
One of the best performances to improve CPT stability and solubility in biological

conditions has been to covalently conjugate it with a β-cyclodextrin (β-CD) derivative

condensed with difunctionalized PEG comonomers, obtaining linear high molecular

weight (over 50 kDa) β-cyclodextrin-CPT based polymers [114-117]. For this purpose, a

CPT-glycine ester derivative was prepared, which was subsequently linked to the β-CD

derivative by amide bond (Fig. 6). This way, the solubility of CPT is increased by more

than three orders of magnitude [114]. Moreover, this nanomedicine showed higher

concentration in tumor and superior efficacy than irinotecan in the treatment of CD-1

female athymic nude mice bearing LS174T human colon carcinoma, HT-29 human

colorectal carcinoma, H1299 human non-small-cell lung cancer model or Panc-1 human

pancreatic cancer cell tumors [115-116,118-119]. As a consequence, a commercial

product (CRLX-101, formerly IT-101) has been released, currently under clinical trials.

Fig. 6. Schematic illustration of the synthesis steps involved in preparing 6A,6D-Diiodo-

6A,6D-dideoxy-cyclodextrin (CDDI) polymerized derivative. (Reprinted with permission
from ref. 114. Copyright © 2003 American Chemical Society)

β-cyclodextrine has also been the aim of further development to improve CPT solubility

and stability. In a relevant study, hollow nanospheres were prepared based on β-

cyclodextrin-grafted α, β-poly(aspartic acid) (β-CD-graft-PAsp). This conjugate favored

the inclusion of CPT and lactone ring stability. In vitro cytotoxicity assays in L929 mouse

19
muscular cell line suggested a decrease of cytotoxicity at higher dose of CPT@β-CD-

graft-PAsp [120].

A different CPT delivery system based in β-cyclodextrine is the nanosponge (CDN)

prepared to improve CPT transport to prostate cancer cells, including androgen-

refractory (DU-145 and PC3 human prostate cancer cell lines) and sensitive (LnCaP

human prostate cancer cells) cell lines. CPT was incorporated in the CDN structure and

its interaction with the nanosponge was confirmed by thermal analysis. Cell proliferation

and clonogenic assays showed a greater responsiveness of CDN-CPT against PC3 and

DU145 cell lines, blocking colony formation and cell growth starting at 1 nM dose.

Besides, in vitro assays against LNCaP cell line, demonstrated that CDN-CPT inhibited

androgen receptor gene expression in androgen-resistance prostate cancer patients at

10 nM, whilst CPT was inactive at similar doses [121].

Poly(L-glutamic acid) (PGA) polymers have been combined with CPT by direct

esterification [122], or by using a short linker, like glycine [123], obtaining loadings of

nearly 40%. The administration of four doses of 7% CPT-loaded PGA by iv injection (one

single dose every four days, with an equivalent CPT dose of 40 mg/kg) over Sprague-

Dawley female athymic nude mice bearing NCI-H322 human lung carcinoma tumors

showed a 32-day tumor growth delay with regards to untreated mice, prolonging the

median survival of treated mice by 4-fold [122]. Moreover, by increasing polymer

molecular weight up to 50 kDa and CPT loading up to 37%, it was reported a tumor

growth was delayed to less than half in comparison to untreated mice after a 30 mg/kg

CPT equivalent dose over athymic NCr (nu/nu) mice bearing NCI-H460 human lung large

cell carcinoma tumors [123].

In the last decade, among different biopolymers, the use of chitosan for drug delivery

has been widespread in order to improve biocompatibility and bioavailability of cytotoxic

compounds [124]. In one of those applications to prepare a CPT delivery carrier,

hydrophobically modified glycol chitosan (HGC) nanoparticles were constructed by

20
chemical conjugation of hydrophobic 5b-cholanic acid moieties to the hydrophilic glycol

chitosan backbone [125]. CPT was easily encapsulated into HGC nanoparticles (CPT-

HGC, 280-330 nm in diameter), which showed sustained release for one week. CPT-

HGC exhibited significant antitumor effects and high tumor targeting ability towards

MDA-MB231 human breast cancer xenografts subcutaneously implanted in nude mice.

Tumor growth was significantly inhibited after iv injection of CPT-HGC nanoparticles at

doses of 10 mg/kg and 30 mg/kg, compared to free CPT at a dose of 30 mg/kg.

More recently, CPT has been conjugated with hyaluronic acid (HA-CPT) with variable

molecular weight, in order to overcome the low solubility and poor targeting ability of the

naked drug [32]. Here, CPT was incorporated using two linkage molecules such as

succinate and adipic acid dihydrazide. The authors studied the influence of the polymer

molecular weight over solubility, drug loading and stability. Solubility assays confirmed

that these conjugates increase CPT solubility, mostly those samples with lower

molecular weight. Furthermore, in vitro efficacy assays demonstrated that the antitumor

activity of HA-CPT polymers were higher than the naked drug against HepG2 human

liver cancer cell line due to an HA receptor overexpression.

A singular and very efficient approach for CPT delivery has been achieved by

encapsulating the drug during self-assembling peptide amphiphile (PA) nanofibers

through a solvent evaporation technique [126]. In vitro efficacy assays against BT-474

human breast cancer cells, MCF-7 human breast adenocarcinoma cell line and SKBR-3

human breast cancer cell line, demonstrated that the intracellular release of the cargo

induced cell death with half maximal inhibitory concentration (IC50) values similar to those

observed with the naked drug. Furthermore, in vivo assays of the PA-encapsulated CPT

in a mouse orthotopic xenograft model (Sprague-Dawley female athymic nude mice) of

BT-474 human breast carcinoma suggested that the nanomaterial delayed

subcutaneous tumor growth.

21
3.2. Inorganic nanocarriers

Currently, a wide variety of inorganic nanoparticles may have biological applications.

They usually show high stability in the biological environment, ease of functionalization

(allowing multifunctionality) and unique physico-chemical properties (optical, magnetic

and electronic) that can be tailored by controlling particle structure, composition, size

and shape. Materials as semiconductor quantum dots, magnetic nanoparticles and

plasmonic nanoparticles, among others, have been proposed for imaging and therapy

[127,128]. However, despite the interest raised by inorganic nanoparticles as DDSs,

most of these systems suffer of biocompatibility issues, mostly related to the toxicity of

some structural components and the strong interaction with serum proteins, which results

in rapid blood clearance and hepatic removal [129]. Moreover, occasionally, the

elimination from organic tissues may be an issue, involving the accumulation in the body

of highly toxic elements. Herein, we present a few cases of simple inorganic

nanoparticles that have found application for delivery of CPT, metal oxides and carbon

nanotubes.

3.2.1. Metal oxide nanoparticles

The most significant use of transition metal oxides for biological purposes are magnetic

processes, such as magnetic resonance imaging (MRI) and magnetic induction

hyperthermia. Typically, paramagnetic oxide nanoparticles show a critical size of around

10-20 nm, which is dependent on the material, have a large magnetic moment and are

stable in physiological fluids [129].

Iron oxide (Fe3O4) hollow monodispersed spheres of about 200 nm mean diameter, and

composed of small primary nanoparticles with a 30-40 nm average size, were

synthesized using a one-pot solvothermal method, and further loaded with CPT by

incubation in a chloroform solution [130]. These nanospheres showed negligible drug

leakage before cell internalization, and exhibited slightly higher cytotoxicity than free CPT

against 786-O, HepG2 and HeLa cell lines, due to the enhanced solubility of the

22
transported drug. Furthermore, non-loaded Fe3O4 hollow spheres showed nearly no

cytotoxicity towards the cells, although there is no report on the stability of these

nanocarriers in in vivo conditions.

Very high CPT loading has been achieved into iron oxide (Fe3O4) superparamagnetic

nanoparticles (SPIONs) with 14 nm average size obtained through an iron co-

precipitation method under alkaline conditions [131]. Formulations containing 13 wt%

CPT were produced by incorporation of the drug to a SPION aqueous solution; this

increased up to 26 wt% CPT by functionalizing SPION surface with a PEG. The high

loading capacity was attributed to the amphiphilic nature of the PEG molecule.

Nevertheless, this did not bring about any improvements in cytotoxic activity, as CPT-

loaded nanocarriers demonstrated about 10-15% lower apoptotic levels over H460 cell

culture than the free drug. This was justified on the basis of CPT slow release profile

from the SPION formulations.

3.1.2. Carbon nanotubes

Carbon nanotubes (CNTs) have recently emerged as a new alternative vehicle for

transporting and releasing therapeutic molecules, due to their high surface areas

[127,132]. However, several concerns relating to pharmacokinetics, biodistribution and

toxicity (mostly by liver and kidney accumulation), must be solved before they can go

into clinical application. In this sense, surface chemical modification becomes a crucial

step for preparation of soluble CNTs and efficient drug incorporation [127].

In an attempt to raise CPT water solubility and antitumor efficacy, non-covalent

supramolecular attachment on multiwall carbon nanotubes (MWCNTs) has been carried

out. Surface oxidation of MWCNTs followed by functionalization with poly(vinyl) alcohol

(PVA) allowed CPT loading via π-π interactions (Fig. 7) [133]. This construct was found

to be very stable in simulated body fluid and, in in vitro testing, it was approximately 15-

fold more cytotoxic than free CPT against MDA-MB231 human breast cancer and

metastatic skin A-5RT3 cell lines.

23
 A

Fig. 7. (A) Schematic depiction of CPT loaded MWCNT-PVA. The figure shows CPT
molecule in red, MWCNT structure in black and polymer PVA in blue. (B) UV spectra of
multiwall CNT-PVA and CNT-PVA-CPT samples with different concentrations.
(Reprinted from ref. 133 with permission of The Royal Society of Chemistry)

Another singular CNT-based material suitable for CPT delivery are glyconanosomes

(GNS). These are water-soluble nanodisks created by supramolecular self-organization

and photopolymerization of diacetylenic-based glycolipids on single wall carbon

nanotubes (SWCNTs) surface, and subsequently used as molecular scaffolds that

determine their shape and topology (Fig. 8) [134]. Experiments against MCF-7 human

breast cancer cells suggested that CPT-loaded GNS-SWCNTs presented higher

antitumor activity than the naked drug (killed more than 65% of MCF-7 human breast

cancer cells while CPT alone affected less than 55% MCF-7 cells), which could be

explained because the interaction of GNS/CPT could interfere with cellular CPT

detoxification mechanism in such a way that intracellular CPT retention time increases,

prolonging the span and release time of the drug.

24
 OH O
OH O
HO O S NH O
O
O HO HN
OH 4N 7
OH H
OH O
10 HN
HN HN HN
O 7 O
O O HN
7 7 7 7 O

7
Hydrophillic Hydrophobic 10
head tail
10
9 9 9 10

O
9
N
8 H

O
9
N
8 H

O
9
N
8 H

Fig. 8. (A) Schematic representations of the self-organization and synthesis of

glyconanosomes (GNS). The inset is a TEM micrograph of SWCNT nanoassemblies and
their idealized representative figures. (B) Schematic representation of the ultrasonication
setup for obtaining GNS. (C) Structural and chemical composition of a GNS. (Reprinted
with permission from ref. 134. Copyright © 2013 American Chemical Society)

3.3. Hybrid nanocarriers

As commented before, fully inorganic systems found strong limitations for biological use,

due to possible compositional toxicity and severe interaction with serum proteins. For

this reason, inorganic nanocarriers are better handled surface modified or combined with

organic components, which provide good stability in physiological fluids, low toxicity and

immunogenicity, and minimized reactions with serum proteins, to control their fate in

biological environment [129]. These composites consisting of two or more constituents

25
distributed in the nanoscale or molecular range are hybrid materials. Typically, the hybrid

nanocontainers are made of inorganic and organic building blocks, presenting unique

properties depending on the spatial and size distribution of their components [135-136].

Actually, efforts have been done to design hybrid devices combining different functions

or strengths of the individual components in a synergic way, or even to compensate for

the weaknesses of one another [137]. Whilst organic component provides good

biocompatibility and better solubility in physiological fluids, the inorganic moiety offers

almost unlimited external surface area to bind different functionalities, improving the

capabilities of the DDS. On the other hand, the main handicap of these systems is the

lack of long-term toxicity studies about the possible interactions within the body of the

different components.

A myriad of compositions and models have been reported with potential use in

nanomedicine. According to their structural configuration, we have distinguished three

different types of hybrid materials with applications as DDS: organic-inorganic

nanoparticles, core-shell systems and metal-organic frameworks. However, in this

section we only present a brief description of some of the most representative examples

of the different systems proposed, as most of these hybrid nanocarriers for CPT delivery

carry molecular devices sensitive to specific physico-chemical stimuli, which act as

triggers for selective release activation at the targeting tissues and cells. These last are

described in detail in the next section.

3.2.1. Organic-inorganic nanoparticles

Organic-inorganic hybrid materials combining the individual advantages of both

components while overcoming their intrinsic drawbacks have received extraordinary

attention in the last decade. In the case of biomedical applications, their complex

chemical composition, their variable structure, but mostly the inherent multifunctional

property, offer plenty of room for the development of novel systems that are able to carry

out associate actions inside the body, as imaging and therapeutics.

26
Mesoporous silica nanoparticles (MSNs) modified with organic ligands are probably the

most widely used inorganic material for hybrid DDS development [138]. These particles

have large surface areas and porous interiors that can be used as reservoirs for storing

hydrophobic drugs as CPT. Moreover, particle surface and pore size can be easily

tailored to selectively store and deliver different molecules. In this context, MSNs were

stabilized in aqueous medium by surface modification with methylphophonate groups,

which reduced aggregation [139]. Then CPT was absorbed within the 2 nm diameter

pores and the particles were incubated with different cells lines (PANC-1 human

pancreatic carcinoma cell line, Capan-1 human pancreatic ductal adenocarcinoma cell

line, AsPc-1 human pancreatic adenocarcinoma cells, SW 480 human colon

adenocarcinoma cell line and MKN 45 human gastric cancer cell line) to determine if they

were able to transport the hydrophobic drug into the cancer cell. In all cases, results

indicated that the cytotoxic efficacy of CPT-loaded MSNs was very similar to that of the

dissolved CPT. In an ulterior study, folic acid was incorporated to the surface of MSNs

(FA-MSNs) as targeting molecule to pancreatic cancer cells. Then, in vivo biodistribution,

therapeutic efficacy and biocompatibility of the CPT-loaded FA-MSN were evaluated in

BALB/cAnNCrj-nu nude mice bearing MCF-7 human breast carcinoma tumors. These

authors showed that nanoparticles were able to repress subcutaneous tumor growth with

a minimum therapeutic dose of about 0.5 mg/kg CPT-loaded FA-MSN for complete tumor

growth inhibition. Inhibition of tumor growth was also complete in the CPT-treated group

but with clear signs of toxicity in blood analysis, as high concentration of liver enzymes,

hypoalbuminemia and hypoproteinemia, elevated creatinine kinase and decreased

levels of calcium and phosphorous. Moreover, one mouse in the group treated with CPT

was euthanized on the 51st day due to manifestation of severe toxicity. Conversely, the

group treated with CPT-loaded FA-MSN only presented mildly elevated blood

concentration of liver enzymes [140-141].

27
In another MSN-based model, MSNs were functionalized with hyaluronic acid (MSN-HA)

to overcome agglomeration issues. The cellular experiments showed that MSNs-HA is

capable of selectively targeting specific cancer cells over-expressing the CD44 protein,

leading to rapid and concentration dependent uptake by the cancer cells through the

receptor-mediated endocytosis mechanism [142]. Subsequently, CPT was encapsulated

in the pores, and the system showed enhanced cytotoxicity to HeLa cells compared to

both free CPT and CPT-loaded MSNs-HA in the presence of excess free HA.

The integration of functional organic fragments into the framework of mesoporous silica

to produce periodic mesoporous organosilica materials (PMOs) was reported in 1999 by

three different groups [143-145], by using bridged organoalkoxysilane precursors

(R’O)3Si-R-Si(OR’)3 (R: organic group). PMOs feature molecularly organic-inorganic

hybrid compositions where the organic R moiety homogeneously distributes within the

whole framework without blocking pore channels. The recent development shift from bulk

materials to nanosized PMOs has resulted in mesoporous organosilica nanoparticles

(MONs) with biomedical applications [146]. As a consequence of this hybridization

strategy the chemical nature of the silica framework can be significantly altered, offering

a number of specific biological effects and functions suitable for biomedicine, as tunable

biodegradation and improved biocompatibility. However, with regards to MSNs, the in

vivo evaluation of the biological effects and the biosafety of MONs is still in its infancy,

and complete studies of biodistribution, excretion, biodegradation, and toxicity are still

pending [147]. In this context, in a singular study, monodisperse MONs co-doped with

fluorescence resonance energy transfer (FRET) cascades composed of fluorophores

anthracene (Anth), naphthalimide (Naph) and biphenyl (Biph) at various ratios were

prepared by a modified Stöber method, and CPT was loaded inside the pores [148].

Next, release experiments in PBS demonstrated that sample containing the three

fluorophores (Biph/Anth/Naph) presented quicker release profile than samples with two

fluorophores (Biph/Anth) or with only Biph. Actually, this CPT tunable retention ability is

28
governed by the quantity of incorporated fluorophores and their polarities, which follows

the order of Naph > Anth ~ Biph. Moreover, although the release rate can be more finely

tuned in an asynchronous way (a single drug) by varying the ratio between fluorophores,

it also possible monitoring the synchronous discharge of several drugs. In this sense, it

can be said that drug releasing performance of MONs can be well correlated with its

color. Studies on the cellular uptake efficiency were carried out on HeLa human cervix

carcinoma cell line, resulting that by varying the ratio between the different fluorophores,

a colorful bioimaging system benefited from FRET should be obtained. Also, the cell

viability evaluated using MTT assay indicated similar growth inhibition potential for MONs

than the free drug.

Fig. 9. Illustrations (A, D, and G), emission spectra (B, E and H) and TEM images (C, F
and I) of monodisperse mesoporous organosilica nanoparticles co-doped with FRET
cascades composed of triple fluorophores: anthracene (Anth), naphtalimide (Naph) and
biphenyl (Biph). The red plus sings indicate the maximum absorption wavelength of
different fluorophores. (Reprinted from ref. 148. Copyright © 2012 American Chemical
Society)

Closely related to MSNs, mesoporous bioactive glass nanoparticles, with higher specific

surface area and pore volume than conventional bioactive glasses, have been recently

29
proposed for hybrid DDS development [149]. In a significant study, the mesoporous

bioactive glass SiO2−CaO−P2O5,(MBG-OH) was functionalized with folic acid and

fluorescein isothiocyanate silanes (MBG-FA) and, subsequently, CPT was absorbed

within the pores (MBG-CPT) [57]. In vitro testing over HeLa human cervix carcinoma

cells, which are overexpressed folate receptors, and L929 murine fibroblast cells, which

are deficient folate receptors, suggested that while loading efficiency was better for FA

free nanoparticles, MBG-CPT showed higher cytotoxicity in HeLa cell line than MBG-OH

because of the increased cell uptake of anticancer drug delivery vehicles mediated by

the FA receptor. Confocal microscopy experiments confirmed that nanoparticles enter

the cells by endocytosis and that cell uptake was mediated by the folate receptor.

Conversely, stable hybrid DDSs can also be obtained by modification of an organic

matrix with inorganic components. In an illustrative example, silane-modified amphiphilic

chitosan was synthesized by anchoring a coupling agent, (3-aminopropyl)triethoxysilane,

to an amphiphilic carboxymethyl-hexanoyl chitosan. Further self-assembly in a CPT

solution led to drug loaded nanovesicles with stable polygonal geometry, consisting of

ordered silane layers of 6 nm in thickness [150]. The hybrid presented well-controlled

encapsulation and release profile for CPT, which are strongly conditioned by chemical

composition and silane concentration. These nanovesicles exhibited excellent

biocompatibility and cellular internalization capability in ARPE-19 human retinal

pigmented epithelium cell line, confirming by confocal microscopy experiments that

nanomaterial entered the cytosolic compartment to release the cargo. Unfortunately, no

cytotoxicity study comparing with the naked drug was provided.

In another organic-inorganic model CPT was first incorporated into micelles derived from

negatively charged biocompatible surfactants, such as sodium cholate, and these

negatively charged micelles were then encapsulated in nanoparticles of magnesium–

aluminum layered double hydroxides (LDHs) by an ion exchange process [151]. The

encapsulation method allowed for an approximately 3-fold increase in CPT solubility.

30
When administered to 9L Glioma cells, the nanobiohybrid containing CPT resulted in

significantly lower survival times compared to untreated cells, or to cells incubated with

the surfactant, the pristine LDH, or water (delivery medium). In addition, LDH surface

modification with bifunctional succinimidyl compounds allows the attaching of active

targeting biomolecules, which may direct these nanohybrids towards specific cells or

subcellular components, increasing their cell uptake ratio. Although these

nanocomplexes showed slightly lower cytotoxic activity than free CPT, they look like a

promising biocompatible model for CPT delivery, allowing drug administration in a dose-

controlled fashion due to the good dispersion of the complexes in water.

3.2.2. Core-shell systems

Complex nanoscopic core–shell architectures are potentially useful for biomedical

applications, for example, as image developers, smart sensor materials or as DDSs

[152]. In this context, the target of designing multifunctional nanoparticle architectures

for biomedical applications has strongly stimulated progress in synthesis of core–shell

nanoparticles with increasing structural complexity. The incorporated functionalities may

provide high resolution imaging and sensing (e.g., using fluorescent molecules,

semiconductor nanocrystals and/or metal nanoparticles), accurate local temperature

manipulation in cancer hyperthermia (magnetic nanoparticles, plasmonic nanoparticles),

and controlled drug delivery and release, combining organic and supramolecular

chemistry with colloidal nanoscience. Here, different models for delivery and controlled

release of CPT have been reported, the most significant of which are described below.

Core-shell organic nanocapsules for oral delivery of camptothecin were formulated with

a core made of amphiphilic cyclodextrins (CDs) and an external coating of chitosan (CS)

[153]. These formulations were in the range of 180-220 nm, with narrow size distribution.

CPT was loaded in the inner polymer and then positively charged chitosan was added

to cover the particles. The resulting nanocapsules were found to be stable in simulated

31
gastrointestinal media and proved favoring CPT permeability through an artificial mucus

gel layer and through Caco-2 (human colorectal adenocarcinoma cells) membrane. In

vivo animal studies in CD1 female mice demonstrated that CPT-loaded CD@CS

nanocapsules promoted drug absorption at the intestinal tract vs stomach uptake. This

could be an interesting vehicle for CPT oral administration, although it does not solve the

important issue of systemic toxicity.

One of the most studied core-shell systems is that obtained by protecting a metal

nanoparticle with an organic coating. In one representative example, multifunctional

nanocarriers of gold nanoclusters as core and folate (FA)-conjugated amphiphilic

hyperbranched block copolymer (based on poly(L-lactide) inner arm and FA-conjugated

sulfated polysaccharide outer arm) as shell, were synthesized for CPT targeted delivery

(Fig. 10) [154]. These nanocarriers presented a release pattern curve at different pH in

two stages, and an initial rapid release (1 h) was followed by a sustained release period

Fig. 10. Schematic illustration of gold nanoclusters as core and folate (FA)-conjugated
amphiphilic hyperbranched block copolymer as shell. (Reprinted with permission from
ref. 154. Copyright © 2012 American Chemical Society)

32
(up to 15 h), and then reached a plateau. Cytotoxicity studies over HeLa cells showed

that, with regards to the free drug, CPT-loaded nanocarrier provided slightly higher

(about 10%) anticancer activity, also gaining specificity to cancer cells with

overexpressed FA receptor moieties.

Fully inorganic core-shell hybrids can also be used for CPT delivery. In a most usual

configuration, the nanocarrier is obtained by coating a metal nanoparticle with a silica

outer layer, CPT being encapsulated inside the silica pores. In this sense, recently, our

group carried out combined chemo and photothermal therapy in in vitro testing by means

of multifunctional nanoparticles formed by plasmonic gold nanoclusters (GNCs) with a

protecting shell of porous silica that contains CPT (GNC@SiO2) [155]. This

therapeuticnanoplatform associated the optical activity of small GNCs (longitudinal

surface plasmonresonance peak, LSPR, at 660 nm) to the cytotoxic activity of CPT

simultaneously released for the efficient destruction of cancer cells. A method was used

for the controlled assembly of 15 nm diameter gold colloids into stable clusters with a

tailored absorption cross-section in the vis/NIR (near-infrared spectroscopy) spectrum

(Fig. 11). Clusters were further encapsulated in an ordered homogeneous mesoporous

silica coating that incorporates CPT molecules within the pores. After internalization in

42-MG-BA human glioma cells, GNC@SiO2 were able to produce effective

photothermolysis under femtosecond pulse laser irradiation of 790 nm. Moreover, the

simultaneous release of CPT during the process increased cell death up to 70%. On the

other hand, control experiments with no irradiation reported 45% cell death for CPT-

loaded GNC@SiO2 nanoparticles and 30% cell death for the free drug. This therapeutic

model could be of interest for application in the treatment and suppression of non-solid

tumors like glioblastoma multiforme.

In a similar way, mesoporous silica-coated gold nanorods (GNR@SiO2) can find

applications in cancer therapy due to their optical properties (LSPR at 770 nm) and

cytotoxic activity (CPT loading in the mesopores). Moreover, tLyP-1, a kind of tumor

33
homing and penetrating peptide, was engrafted to GNR@SiO2 [156]. The obtained

GNR@SiO2−tLyP-1 was loaded with CPT and tested over hMSC human mesenchymal

stem cells, HeLa human cervix carcinoma and MCF-7 human breast cancer cell lines.

After cell internalization, core-shell nanorods were irradiated by NIR illumination. Then,

all cells were killed instantaneously by the increase in temperature caused by

GNR@SiO2−tLyP-1 surface plasma resonance and CPT cytotoxic activity. Moreover, the

systematic toxicity of CPT on human mesenchymal stem cells was minimized, because

the GNR@SiO2−tLyP-1 selectively targeted and penetrated into the tumor cells, and little

hydrophobic CPT was released into the culture medium or blood.

Fig. 11. (A) Schematic of synthesis and controlled aggregation of gold colloids protected
with a thin silica layer into gold nanoclusters (GNC@SiO2). (B) UV-vis/NIR absorbance
spectra of gold nanoclusters obtained at different NaOH concentration. (C) Photograph
of the different colloidal gold dispersions. (D) Fluorescent microscope images of 42-MG-
BA human glioma cells incubated with GNC@SiO2 (with and without CPT) after laser
irradiation. A control with no nanoparticles (NP) is also shown. (Adapted from ref. 155
with permission of The Royal Society of Chemistry)

34
3.2.3. Metal organic framework nanoparticles

Due to their porous ordered structure and virtually infinite combinations of metals and

ligands, metal-organic frameworks (MOFs) can exhibit exceptionally high surface areas

with large pore sizes. Consequently, they have been recently proposed for applications

in loading and controlled release of several drug molecules [157]. However, MOFs need

to be scaled down to the nanoregime to form nanoscale metal-organic frameworks

(NMOFs) to be used as delivery vehicles for imaging agents and drug molecules [158].

NMOFs possess some potential advantages over existing nanocarriers given their

compositional and structural diversity, which allows for the synthesis of different shapes,

sizes and chemical properties, and their biodegradability, a result of relatively labile

metal-ligand bonds. Surprisingly, despite the huge potential of these nanomaterials for

novel DDSs development, stability in physiological fluids determination and identification

of potential hazards associated to their components are still a matter to investigate [159].

Very few toxicological studies concern NMOFs, usually limited to specific cell lines, which

makes it difficult to compare the results obtained. In this context, there are very few

examples of MOF application to CPT delivery yet, most of them focused on the structure

of ZIF-8 [160,161].

A general synthetic route was optimized to encapsulate several small molecules,

including fluorescein and CPT in monodisperse zeolitic imidazolate framework-8 (ZIF-8)

uniform of 70 nm particles with single-crystalline structure (Fig. 12) [160]. Evaluation of

fluorescein-encapsulated ZIF-8 nanospheres in MCF-7 human breast adenocarcinoma

cell line demonstrated cell internalization and minimal cytotoxicity. The 70 nm particle

size facilitates cellular uptake, whereas ZIF-8 framework easy dissociation results in

quick endosomal release of the small-molecule cargo. Here, it was shown that

encapsulated CPT ZIF-8 particles show enhanced cell death, which involves cell uptake

and intracellular release of the drug.

35
Fig. 12. Schematic illustration of MOF application for small molecule delivery (fluorescein
in green (A) and CPT in blue (B)) inside of ZIF-8. CPT molecule fits well within the
microporous framework, which loads a maximum of 2 % drug. (Reprinted from ref. 160.
Copyright © 2014 American Chemical Society)

Overall, the incorporation of CPT to a nanocarrier definitively improves its therapeutic

efficacy. All vehicles provide a clear increase of drug stability, as the encapsulated or

bonded molecule is protected from enzymatic degradation and lactone ring cleavage

during its transit through blood stream and tissues to tumor location. Moreover, colloidal

stability of these formulations can boost drug solubility several orders (e.g., β-

cyclodextrins), which gathered to the stability rise improves the bioavailability. However,

the most significant effect of incorporating CPT into a nanocontainer is the dramatic

toxicity reduction and therapeutic window extension, as the transported molecule has

very little activity before it is discharged at the tumor place. As a consequence, the in

vivo antitumor activity increases with regards to the free drug, due to an improved

bioavailability and the possibility to increase the dose with no side effects. In addition,

many of these nanomedicines offer the possibility of delivering several drugs in a row,

which is very important for the treatment of MDR cancer, and allow for the incorporation

of targeting molecules that favor selective therapy. Finally, inorganic and hybrid materials

can present unique properties (e.g., optical activity, magnetism, semiconductor activity),

that enhance the potential of these systems in therapeutics and diagnostic.

36
4. Stimuli responsive systems

These are currently the most representative DDSs in Nanomedicine. The main

characteristic is their ability to deliver a therapeutic agent into the target cells with no

previous release. This is due to the accurate control of the release process through a

specific intracellular stimulus, which minimizes undesired side effects. Stimuli-

responsive nanocarriers can be classified in endogenous or exogenous, depending on

the stimulus which activates the drug release mechanism. Examples of endogenous

stimuli are pH, redox potential and enzyme activity. On the other hand, classical

exogenous stimuli include magnetic fields, ultrasounds, light and temperature [162-165].

4.1. pH-sensitive systems

Several pH-sensitive DDSs have been developed based on the pH difference between

normal and tumor tissue. Here, some of these platforms take advantage of the fact that,

in some cases, tumor tissue pH profile is slightly lower than normal tissue. However, the

heterogenicity of extracellular pH both within and between tumors poses a significant

challenge, as low extracellular pH regions are likely to be less accessible to nanoparticles

that do not distribute readily within the tumour interstitium. This factor, together with the

lower pH values inside endosomes, suggests that enhancing endosomal escape is the

most realistic objective for pH-sensitive nanocarriers in the short term [166].

Polymeric, pH-responsive micelles have been prepared by a solvent evaporation

method. For this purpose, hydrophilic methyl ether poly(ethylene glycol) (MPEG) and

biodegradable poly(β-amino ester) (PAE) were copolymerized to give MPEG-PAE block

copolymer (Fig. 13). Then, CPT was encapsulated into the micelles (CPT/MPEG-PAE)

through hydrophobic interactions between drug and PAE. At pH 6.4-6.8 CPT/MPEG-

PAE were demicellized and the encapsulated CPT was rapidly released from these

nanocarriers, although under pH 7.4 the release rate was clearly slower. In vitro

cytotoxicity assays in MDA-MB231 human breast tumor cells confirmed that these

37
micelles provoked severe cytotoxicity within an acidic environment due to rapid CPT

release, whereas cytotoxicity decreased at physiological pH [167].

Fig. 13. (A) Chemical structure of methyl ether poly (ethylene glycol) - poly(β-aminoester)
block copolymer (MPEG-PAE). Letters x and y indicate the number of times that the
block polymer is repeated. (B) Schematic diagram of pH-responsive CPT/MPEG-PAE
micelles at weakly pH condition. The figure shows CPT in blue, and in red and yellow
the MPEG-PAE block copolymer. (C) Size distribution of MPEG-PAE micelles in PBS at
pH 7.4 by dynamic light scattering (inset: TEM image). (Reprinted from ref. 167,
Copyright © 2010, with permission from Elsevier)

Another pH-sensitive nanoscaled system for CPT delivery has been obtained by

conjugation to a biocompatible, hydrophilic copolymer of mucic acid and polyethylene

glycol (MAP). When this polymer-drug conjugate was placed in water, it self-assembled

into MAP−CPT nanoparticles of ca. 30 nm [168]. Then, antibody herceptin was

complexed with MAP-CPT to form a targeted nanoparticle. A strong dependence of

release rate with pH was observed but, unlike the standard behavior, as pH increased

from 6.5 to 7.4, the release half-live dropped from 338 to 58 h for MAP−CPT

nanoparticles. In vitro testing over BT-474 and HER2 human breast cancer cell lines

showed that cellular uptake of nanoparticles was enhanced by 70%, compared to non-

targeted version, by the incorporation of a single herceptin antibody molecule per

nanoparticle. Moreover, CPT was released from MAP-CPT nanoparticles by hydrolysis

and nanoparticle disruption by fat. The targeted MAP-CPT nanoparticle system carried

38
ca. 60 CPT molecules per nanoparticle and showed prolonged plasma circulation with

an elimination half-life of 21.2 h and AUC value of 2766 μg h/mL at a 10 mg CPT/kg tail

vein injection in female BALB/c nude mice.

Encapsulation of CPT in micelles with a hydrophilic shell of chitosan and a hydrophobic

core of poly(p-dioxanone) has been also used to control drug release on the basis of

environment pH changes [169]. CPT was incorporated in the core of this system and its

release was triggered at pH 7.4, 6.2 and 5.0. In vitro drug release studies in HeLa human

cervix carcinoma and L929 mouse fibroblast cells demonstrated that the micelles

presented a much faster release of CPT at pH 5.0 than at pH 7.4, whereas blank micelles

without CPT were found to be nontoxic in both cell lines. The good internalization effect

of these micelles was explained by the electrostatic interactions between positively

charged micelles and cell membranes.

In another recently published study, an inorganic pH-sensitive nanocarrier was able to

deliver different chemotherapeutic drugs simultaneously for a better synergetic effect.

This nanoplatform was based on hollow silica nanoparticles sealed with ZnO quantum

dots (QDs). Subsequently, CPT was encapsulated in the hollow core through

hydrophobic interactions. On the other hand, doxorubicin (DOX) was deposited on the

mesoporous shell via electrostatics interactions with carboxylate groups incorporated on

the mesoporous surface [170]. This cooperative drug loading largely increased drug

encapsulation efficacy of both CPT and DOX up to 89.28% and 44.98%, respectively.

Drug release was compared at pH 7.4 and pH 5.0, concluding that in acid medium the

amount of discharged drugs was higher due to ZnO QDs disintegration.

4.2. Redox-sensitive systems

It is well-known the difference in redox potential between the oxidizing extracellular

space and reducing intracellular space, which is related with glutathione (GSH)

39
concentration. This fact has found application in the building of specific DDSs able to

selectively discharge their therapeutic load after cell uptake. Here, drug covalent linking

to nanocarriers is mostly carried out by disulfide bridge, which makes possible to design

of different sensitive systems. The main concern for in vivo application of these systems

is the presence of GSH in serum which, despite the fact that its concentration is less than

one order below that of cytosol, may provoke some premature release during CPT transit

at blood stream [171].

Hybrid micelles subjected to redox-responsive cleavage have been developed by cross-

linking CPT-loaded organic units with disulfide bonds [172]. These micelles were

obtained via coprecipitation of disulfide-containing CPT-poly(tyrosine(alkynyl)-O-

carboxyanhydride) conjugate and monomethoxy poly(ethylene glycol)-b-

poly(tyrosine(alkynyl)-O-carboxyanhydride), followed by cross-linking of the micellar

core via click chemistry (Fig. 14). In this case, CPT was rapidly released due to the

elevated concentration of cytosolic glutathione that disorganizes micelle structure by

disulfide bond cleavage, which is present both in the CPT prodrug and the cross-linker.

In vitro cytotoxic assays against MCF-7 human breast cancer cells suggested high

anticancer activity due to the disassembly of the cross-linked micelle and rapid CPT

release.

Also recently, our group implemented a new synthetic strategy by direct coupling of as-

synthesized (pyridin-2-yldisulfanyl)alkyl carbonate derivatives of CPT with thiol groups

of silica hybrid nanoparticles containing a non-porous core and a mesoporous shell [173].

Upon reaction with thiols in physiological conditions, disulfide bridge cleavage occurs,

releasing the naked drug after an intramolecular cyclization mechanism (Fig. 15).

Additional incorporation of a fluorophore into particles core facilitated imaging at the

subcellular level for the monitoring of uptake and delivery. Confocal microscopy

experiments in HeLa human cervix carcinoma cells confirmed that nanoparticles enter

40
Fig. 14. Schematic procedure for preparation of redox-responsive uncross-linked (UCL)
and core-cross-linked (CCL) hybrid micelles. (Reprinted partially with permission from
ref. 172. Copyright © 2013 American Chemical Society)

the cells by endocytosis but are able to escape from endo-lysosomes and enter the

cytosolic compartment to release their cargo by glutathione reducing activity. Moreover,

in a separated study [174], it was shown that prodrug side chain carbon number (n)

determines material hydrophobic properties and, as a consequence, its stability in

aqueous medium and cell uptake kinetics. When n value increases, the negative surface

charge decreases dramatically due to a shielding effect provoked by hydrophobic

ligands, which promotes particle aggregation and favors cell internalization.

Furthermore, n value also determines the type of products released and, subsequently,

the cytotoxic activity. Although full disulfide bridge reduction occurs in all cases within

the cell, the subsequent intramolecular cyclization releasing CPT, CO2 and the

corresponding thiolactone only happened quantitatively for n=1 (Fig. 15), whereas higher

homologues released low CPT quantities (5% for n=2 and traces for n=3).

41
 mesoporous silica
amorphous silica core
shell with CPT
with Rhodamine B

O O

Si S O O O
S
n
O N
n=1,2,3

HS N
GSH
Si

O O

O O O O
HS Intramolecular
n cyclization N
O N N

O CPT
O
N O n=1: 100 %
O HO
n=2: ~5 %
S
n=3: <1 %
n

Fig. 15. Redox-responsive nanoplatform for CPT delivery by direct coupling of (pyridin-
2-yldisulfanyl)alkyl carbonate CPT derivatives with thiol groups of silica hybrid
nanoparticles containing a non-porous core and a mesoporous shell. Upon reaction with
thiols in physiological conditions, disulfide bridge cleavage occurs, releasing the naked
drug after an intramolecular cyclization mechanism. (Adapted from ref. 173, with
permission of The Royal Society of Chemistry)

A supramolecular strategy was reported to directly assemble the hydrophobic CPT

molecule to a β-sheet-forming peptide sequence derived from the cysteine-containing

au protein through the reducible disulfylbutyrate (buSS) linker, obtaining discrete, stable,

well-defined nanostructures with a high and quantitative drug loading [175]. Drug content

was precisely controlled using the two amine functionalities of the amino acid lysine to

create branching points that allow the attachment of one, two or four CPT molecules,

corresponding to respective drug loadings of 23%, 31% and 38%. Depending on the

number of CPT molecules in the supramolecular design, the resulting nanostructures

can be either nanofibers or nanotubes. Such nanostructures provide CPT protection from

the external environment. Release profiles showed that glutathione degraded the

designed buSS linker, releasing a mixture of intermediates and CPT. Under tumor-

relevant conditions, these drug nanostructures released the active form of CPT and

showed in vitro efficacy similar to free CPT against MCF-7 human breast cancer cells

and 9L and F98L rat gliosarcoma cell lines. In addition, to avoid the formation of

42
intermediates, the same researchers prepared these CPT-nanostructures using a

(disulfanyl)ethyl carbonate linker, that after disulfide bridge reduction by glutathione

followed a self-immolative process to release free CPT, improving cytotoxicity over MCF-

7 cell line [176]. Furthermore, in vivo preliminary experiments, after intratumoral injection

with 36% CPT loaded sample over 6-8 week female BALB/c mice bearing subcutaneous

CT26 murine colorectal adenocarcinoma, showed higher enhanced retention time of

CPT nanotubes in the tumor site than the free molecule, which allows a sustainable

release over a long period [177]. Conversely, no improvement in tumor targeting was

detected with the nanomedicine in case of administration by tail vein, so the potential of

this impressive nanoconjugate as DDS is still to be confirmed.

4.3 Enzyme-sensitive systems

In these drug delivery nanoplatforms, the therapeutic molecule is engaged to

nanoparticles by a covalent bond that may be cleavage by enzymatic activity. Enzyme-

responsive nanomaterials facilitate the targeting of specific tissue by programming drug

release via enzymatic digestion of the nanocarrier or linking moiety [178]. The biological

recognition of the substrate by the enzyme leads to the selective and specific triggering

event to release the cargo, which minimizes toxicological issues related to CPT. In this

context, due to the high concentration of carboxylesterases in the cytosol, most of these

models have been developed over the basis of an ester bond at 20-OH position between

CPT and the nanostructured surface. On the other hand, there are two main

shortcomings in the use of enzyme-responsive devices for CPT delivery: i) the presence

of carboxylesterases in plasma may motivate some nonspecific release outside the

tumor cells, and ii) the binding of CPT to a nanoparticle surface can limit the accessibility

to enzyme active site due to steric effect, slowing down the release process, which

reduces the cytotoxic activity [179].

43
In a singular approach, CPT molecules were directly grafted to carboxyl groups of poly(L-

glutamic acid) (CPT-PGA) through ester bond [180]. Subsequently, a supramolecular

nanoparticle system was obtained by conjugation of CPT-PGA with several polymeric

derivatives (Fig. 16). This system released CPT under physiological conditions by means

of esterase-mediated hydrolysis. Cell viability assays in MCF-7 human breast cancer

cells indicated nanoparticle delivery system IC50 was 4 times that of the free drug,

probably due to the slow release of CPT from polymer. However, a positron emission
64
tomography study with Cu-labeled nanoparticles in C57Bl/6 mice bearing Lewis lung

carcinoma tumors revealed significant tumor specific uptake of nanoparticles. Moreover,

in tumor reduction/inhibition studies these nanomedicines showed much higher efficacy

than the free drug, delaying tumor growth with no visible body weight or any other side

effect.

Fig. 16. Schematic representations of the self-assembly and synthesis of

supramolecular nanoparticles by conjugation of CPT-grafted poly(L-glutamic acid) with
different polymeric derivatives. Legend: Ad-PEG=poly(ethylene glycol); CD-PEI=
cyclodextrin-poly(ethylenimine); Ad-PAMAM=poly(amido)amine (Reprinted from ref.
180, Copyright © 2012, with permission from Elsevier)

Conversely, CPT conjugation to nanoparticles by ester bond is usually performed in two

steps. First, a prodrug is prepared by esterification with a short linker and then, this

44
prodrug is covalently linked to a supramolecular structure. For instance, to incorporate

CPT into PAMAM, CPT was firstly substituted at 20-OH position with glycine through

ester bond and, afterwards, the prodrug was reacted with PAMAM G4, to give PAMAM-

CPT [181]. This material was stable in plasma and the presence of proteolytic enzymes

such as cytosolic esterases promoted conjugate cleavage and CPT release. Cell

internalization and in vitro assays of this nanomedicine in HTC-116 human colorectal

cancer cells indicated a high cytotoxic effect due to nuclear fragmentation and formation

of apoptotic bodies.

As a different model, our group recently prepared a hybrid organic-inorganic silica

nanoplatform for CPT delivery based on enzyme-triggered release. For this sake, CPT

was covalently linked to nanoparticles through an ester bond with the 20-hydroxy moiety,

in order to stabilize its lactone ring and to avoid unspecific release of the drug [179]. The

obtained material was highly stable in plasma, with less than 5% release of the cargo at

physiological pH. Cell internalization and in vitro efficacy assays against HeLa, U87-MG

human primary glioblastoma cell line, HCT-116 human colon carcinoma and HT-29

human colorectal adenocarcinoma cells, demonstrated that nanoparticles carrying CPT

(SNP-CPT) entered cells via endocytosis and the intracellular release of the cargo

induced cell death with half maximal inhibitory concentration (IC50) values and cell cycle

distribution profiles similar to those observed for the naked drug. Furthermore, in vivo

biodistribution, therapeutic efficacy and biocompatibility of the SNP-CPT were evaluated

in hsd:athymic nude-Foxn1 mice bearing HT-29.Fluc subcutaneous tumors, showing

that, although SNP-CPT tended to accumulate in organs of the reticulo-endothelial

system, nanoparticles delayed the growth of subcutaneous tumors while significantly

reducing the systemic toxicity associated to CPT administration (Fig. 17).

45
 a
A 1200
PBS Tumor Volume
CPT
1000 SNP-CPT

(mm3) (Mean ± SEM)

Tumor Volume
800

600

400

200

0
1 3 5 7 9 11 13 15 17 19 21 23
Time of Treatment (days)

B D1 D4 D11 D15 D22

Vehicle

3.0

×109 BLI Intensity (ph/s)

2.5

2.0

CPT 1.5

1.0

0.5

SNP-CPT

Fig. 17. Tumor growth delay in hsd:athymic nude-Foxn1 mice bearing HT-29.Fluc
subcutaneous (s.c.) tumors. (A) Comparative analysis of the localized s.c. growth of the
HT-29.Fluc tumors untreated (PBS) and treated (CPT or SNP-CPT) in hsd:athymic nude-
Foxn1 mice by external measurement (caliper) of tumor volume. (B) In vivo monitoring
of a representative mouse from each treatment group shows radical tumor growth
reduction with regards CPT and the control group. (Reprinted partially from ref. 179,
Copyright © 2011, with permission from Elsevier)

4.4. Light-sensitive systems

In addition to chemically-triggered delivery systems, remote activation of nanoparticles

by light for biomedical applications has become an important field of research [182]. This

type of stimuli-responsive nanoplatforms present a component sensitive to ultraviolet

46
(UV), visible (Vis) and/or near infrared (NIR) light which is able to activate drug release

under irradiation. As an exogenous stimulus, the main advantage of light over

endogenous processes is the possibility to accurately modulate stimulation that controls

drug release. In this sense, light is not only noninvasive but also controllable both

spatially and temporally, allowing for greater safety and specificity [183]. Conversely, low

penetration ability and predominant use of UV and visible light limit the clinical potential

of these systems. So far, many different systems have been described, involving

photocleavage, photodissociation, photoisomerization, photorelease, photothermal,

photo-plasmonic heating, and up-conversion photoisomerization [184].

In a pioneering work, a nanoimpeller-controlled mesostructured nanoparticles was

developed to deliver and release CPT into living cells upon light activation [185]. These

light-triggered MSNs were functionalized with azobenzene moieties in the pore interiors,

with one end attached to the pore walls and the other end free to undergo

photoisomerization. The impeller in the nanopores trapped drug molecules in the dark,

and release them in response to UV light (413 nm) excitation (Fig. 18). In vitro studies in

PANC-1 human pancreatic and SW480 human colon cancer cell lines showed the

photoresponsive behavior of the impeller inside of cells. After 3 h of incubation in the

dark, the cells were irradiated with ~0.1 W cm-2, 413 nm light, for various excitation times

(0 to 10 min). Cell death was induced under photocontrol. In the absence of light

excitation, the CPT remained in the particles and cells were not damaged. However,

illumination promptly expelled CPT from the particles, causing cancer cell apoptosis,

which is demonstrated by nuclear fragmentation and chromatin condensation.

Unfortunately, the application of UV/Vis light in biomedical studies is mostly limited to

proof-of-concept, because irradiation at this wavelength can damage cells and does not

penetrate deep inside the tissues. However, using two-photon excitation (TPE) in the

NIR region instead of UV/Vis light leads to better tissue penetration, lower scattering

47
Fig. 18. Schematic illustration of the light-activated mesostructured silica nanoparticles
functionalized with azobenzene derivatives in the pore interiors. (Adapted with
permission from ref. 185. Copyright © 2008 John Wiley and Sons)

losses and three-dimensional spatial resolution. For this sake, the former nanoimpeller-

controlled MSNs device was functionalized with a high absorption cross-section two-

photon fluorofore, suitable for Forster resonance energy transfer (FRET) to

photoizomerize azobenzene moieties in the NIR region [184]. The two-photon absorption

properties of the fluorophore were retained in the materials and no decrease of cross-

sections σ2 was noticed after encapsulation. The nanoimpeller groups pending in the

porous framework allow the physical entrapment of CPT, which is then kicked out of the

pores by two-photon-triggered photoisomerization. Therefore, the nanoimpellers were

loaded with CPT and then screened under TPE in MCF-7 human breast cancer cells.

Cells were incubated with nanoparticles and further irradiated in at very short time with

a focused laser beam (input 3 W, output 900 mW cm-2). The MTT assay was performed

two days after irradiation. Nanoimpellers loaded with CPT and high

fluorofore/azobenzene ratio significantly increased cancer cell death under TPE in

comparison to no irradiation.

Another representative light-responsive prototype was developed as a self-deliverable

form of nucleic acid-CPT nanostructure (DNA-CPT) that is composed almost entirely of

payload molecules [186]. To tackle this strategy, three CPT molecules were attached to

48
a phenol group connected to a photolabile 2-nitrobenzyl ether moiety. This photolabile

group was linked to a DNA strand to obtain an amphiphile, which self-assembled in DNA-

CPT nanostructure. Under UV irradiation (365 nm), the 2-nitrobenzyl was cleaved,

releasing the DNA from the conjugate and leaving a decapped self-immolative drug core,

which, afterwards, underwent a rapid, spontaneous and irreversible degradation process

to release free CPT molecules. In vitro efficacy assays in SK-BR-3 human breast cancer

cell line confirmed that cytotoxicity of DNA-CPT nanostructure increased significantly by

light triggering, achieving IC50 values similar to those observed with the naked drug.

In a different strategy, the surface of MSNs may be modified in order to deposit a

hydrophobic layer able to monitor drug delivery by adjusting the wetting of particle

surface. In this context, a light-sensitive release system based on a hybrid with MSN

core and spiropyran and perfluorodecyltriethoxysilane coating was built [187]. Particles

are protected from being wetted by water at the optimal ratio of spiropyran to fluorinated

silane (0.249:1), which successfully inhibits drug release. Upon irradiation with UV light

(365 nm), the conformational conversion of spiropyran from a “closed” state to an “open”

Fig. 19. Schematic presentation of the light-sensitive release system based on

mesopororus silica nanoparticles surface functionalized with spiropyran and
perfluorodecyltriethoxysilane in an optimal ratio. Under UV irradiation at 365 nm,
spiropyran was converted to the hydrophilic form, which resulted in the wetting of surface
of MS and the release of trapped molecules diffusing from the penetrated water.
(Reprinted with permission from ref. 187. Copyright © 2014 American Chemical Society)

49
state caused the surface to be wetted, leading to the release of encapsulated CPT from

the pores (Fig. 19). In vitro experiments in EA.hy926 human somatic cell hybrid and HeLa

human cervix carcinoma cells demonstrated an accurate drug controlled release. Here,

CPT loaded nanoparticles (10 mg/g) were incubated in cells for 24 h and then, upon UV

light irradiation a noticeable loss of cell viability was observed with regards to non-

irradiated cell cultures or with no nanoparticles.

4.5. Magnetic-sensitive systems

Magnetic gradients may be used to target therapeutics to specific tissues, while

alternating magnetic fields produce frequency-dependent effects at the nanoparticle

level. This type of nanocarriers implicates para-magnetic or super-paramagnetic

materials which are embedded into organic polymeric moieties, allowing the release of

the drug under an external magnetic stimulus. The inorganic component is designed to

impart responsivity to external magnetic fields, both static and dynamic, in order to

impose a remote magnetic control of drug cargo release. Furthermore, soft matter

architectures provide a very convenient structural scaffold for insertion of therapeutic

principles [188]. Nevertheless, as commented before (section 3.2), the most serious

concern about the use of magnetic nanoparticles within therapeutic formulations is their

biocompatibility, especially in the long term. In particular, their stability when introduced

within the body is a crucial aspect, as the proper dispersion and circulation of

nanoparticles strongly affect drug release kinetic, accumulation, and toxicity. Actually,

SPIONs are the only engineered inorganic nanoparticles approved for human use.

One of the most illustrative drug delivery models of hybrid magnetic materials is based

on MSN pore capping with iron oxide nanoparticles [189]. This may be preferentially

done through chemical bonding. For this sake, MSNs surface was funtionalized with 3-

50
aminopropyltrimethoxy silane groups. Afterwards, CPT was filled into the pores and

these were covalently capped through amidation of the 3-aminopropyl groups at surface

with meso-2,3-dimercaptosuccinic acid functionalized superparamagnetic iron oxide

nanoparticles [190]. The Fe3O4 nano-caps formed a dense and uniform layer tightly

bound to the MSN surface (MSN@Fe3O4). Under magnetic stimulus, the cumulative drug

release was very quick, and prolonged even when the stimulus was removed, due to

detachment of some Fe3O4 nanoparticles (Fig. 20). Such a magnetically-induced

removal of the nano-caps implies a cleavage of the chemical bond between the Fe3O4

nanoparticles and silanized MSNs. The energy induced from the magnetic stimulus over

various timespans is sufficiently large to cleave the chemical bond, resulting in an

effective removal of the nano-caps. Actually, the rate of nano-cap removal and drug

diffusion are a function of the magnetic energy. Then, cytotoxicity studies in A549 human

lung adenocarcinoma epithelial cell line by MTT assay, indicated that nano-Fe3O4-

Fig 20. Schematic illustration of the synthesis and structure of mesoporous silica
nanoparticles capped with iron oxide nanoparticles. The structure was obtained by
capping mesoporous silica nanoparticles (MSN) with monodispersed Fe3O4
nanoparticles through chemical bonding. Under magnetic stimulus, some of the Fe3O4
nanoparticles were removed from the surface of the MSN, and CPT was released. On
the right, a brief illustration of all different ligands used in the synthesis of this material.
(Reprinted from ref. 190 with permission of The Royal Society of Chemistry)

51
capped MSN effectively constrained the CPT from free elution and were well tolerated,

resulting in a cell viability of more than 80%. However, upon magnetic stimulus, about

42% of the cells were killed, and the major cause of cell death was the CPT released

from the MSN@Fe3O4.

Magnetic stimulus can also be used to accelerate drug release from core-shell hybrid

nanoparticles constructed by self-assembly of iron oxide in the presence of polyvinyl

alcohol. Then, CPT was incorporated to the organic component of the core, followed by

coating with a thin layer of silica to eliminate undesirable drug leakage [191]. Here, the

operation of an external magnetic stimulus allows a pulse-type drug release to be readily

achieved in a real time responsive manner without undesirable delays in dosing

accuracy. The intracellular release of CPT from nanocarriers in A549 cells was

stimulated by high frequency magnetic field (HFMF, 2.5 kA m-1, 50 kHz) for 30-120 s.

Upon magnetic stimulus, CPT release profile for the nanocarriers in different eluting

media indicated that the rate of drug diffusion was considerably increased, which is due

to thermally induced disruption of particle nanostructure [192]. Subsequently, cells were

incubated for 18 h, and the anticancer effect was measured by MTT assay. It was found

that CPT IC50 value measured on A549 human lung adenocarcinoma epithelial cells was

around five times lower than that of the free drug when CPT was loaded into magnetically

sensitive nanocarriers and released intracellularly. Moreover, a similar model was

performed by preparation of embedded Fe3O4 nanoparticles in a PLGA core containing

CPT, protected by a lipid–polymer hybrid shell [193]. In vitro testing of these drug delivery

capsules in MT2 mouse breast cancer cells showed significantly suppressed cancer cell

growth upon magnetic field activation.

52
4.6. Other stimuli-responsive systems for camptothecin delivery

In addition to the different stimuli-responsive systems described, in some recent studies

other analogue entities have been proposed for CPT delivery and controlled release

under endogenous or exogenous stimulation. We here summarize some of most

advanced approaches.

Thermo-responsive nanomaterials are a kind of adaptive nanoparticles sensitive to

environmental temperature changes that may help to monitor spatial delivery of a

therapeutic payload. Under pathological local high temperature records (for instance,

those reported in inflammation and cancer) these systems undergo a phase transition

and conformational change that compromise the integrity of the nanostructure and

activate payload release. At this point, polymers can be designed in such a manner so

that this phase transition, which is termed as the lower critical solution temperature

(LCST), takes place at around 37 ºC. Under LCST the hydrogel nanopreparation is

soluble for in vivo injection, but becomes insoluble and aggregated in the tumor regions

heated above its LCST, leading to local drug discharge [194]. In this context, CPT

thermo-sensitive DDSs can be prepared based on spherical core-shell nanoscale

micelles. Firstly, two kinds of thermo-sensitive poly(N-isoproplacrylamide) (PNIPAM)

four-armed star multiblock copolymers were synthesized by atom transfer radical

polymerization. Then, the multiblock copolymers could spontaneously assemble into

regular spherical core–shell nanoscale micelles of about 100-120 nm diameter and LCST

of about 33-35 ºC [195]. Drug release was thermo-responsive, accompanied by

temperature-induced structural changes of the micelles. At 37 ºC, CPT release from

micelles is clearly quicker than at 25 ºC (respectively, 74 and 53% at 48 h) due to

temperature-induced structural alterations of multiblock copolymers. This accelerated

drug release above the LCST is correlated with the enhanced hydrophobicity of PNIPAM

shells, which leads to micelle shrinkage, and makes CPT diffuse out quickly. In vitro

cytotoxicity studies carry out by MTT assay over MDA-MB231 human breast cancer cell

53
line, demonstrated slightly lower activity for the CPT-loaded micelles than for the free

CPT, probably due to time-consuming CPT release from the micelles.

Ultrasounds represent another effective method for attaining spatio-temporal controlled

release at the target cells, preventing side effects to healthy tissues. Ultrasound waves

can trigger drug through the thermal and/or mechanical effects generated by cavitation

phenomena or radiation forces. Here, physical forces associated to cavitation can induce

nanocarrier destabilization, drug release and transient increase in vessel permeability,

leading to the cellular uptake of therapeutic molecules. Acoustically active

perfluorocarbon nanoemulsions have been proposed for CPT encapsulation, in order to

circumvent its well-known bioavailability issues as insolubility, instability and toxicity

[196]. The nanoemulsions were firstly prepared with perfluoropentane, perfluorohexane

and coconut oil as the core of the inner phase, which incorporates CPT molecules. Then,

a stabilizing coating made of phospholipids and/or Pluronic F68 was applied, obtaining

a mean droplet diameter of 220-420 nm. For the study of ultrasound influence on CPT

release the nanomulsion was exposed to ultrasound using a 1 MHz probe with a 1.5

W/cm2 intensity for 2 h. However, CPT released from the nanoemulsions was limited,

with less than 15% of the drug being discharged over 48 h. Moreover, it was found that

droplet diameter had an influence in release rate, as the amount of exposed surface

decreased for bigger droplets, which slows down CPT liberation. Furthermore, in order

to simulate parenteral administration conditions, plasma was added to the

nanoemulsions and, next, sonication with ultrasound energy was used to stimulate the

liquid-to-gas transition of perfluorocarbons [197]. As the droplets became gas bubbles,

they began to oscillate or resonate and, eventually, bubbles were destroyed and

provoked a local drug release. Unfortunately, no biological study is reported with this

interesting CPT controlled release model.

Recently, glucose-responsive materials have attracted research interest due to their

potential application as DDSs. Most of these systems have been developed as platforms

54
for glycemia control in diabetes mellitus patients. Among them, the most studied model

is that based on phenylboronic acid [198]. In aqueous medium, phenylboronic acid holds

an equilibrium between an uncharged form and a charged form. The first one is

hydrophobic but the second form is hydrophilic. This last is able to form a stable

phenylborate with cis-diol compounds, like glucose, which shifts the equilibrium in the

direction of increasing the hydrophilic form. Usually, this shift is enough to increase the

swelling degree of crosslinked nanogels or to disintegrate self-assembled micelles,

causing the entrapped payload to be released into solution. This provides a way for

designing polymeric carriers for self-regulated delivery not only of insulin but also of other

therapeutic agents. For the purpose of CPT delivery, hybrid nanoparticles with iron oxide

and mesoporous silica were loaded with the drug and further capped with a

phenylboronic-dextran conjugate [199]. In a further step, CPT was encapsulated in silica

mesopores followed by incubation with dextran (Mw 6000 and 100000), which bounded

with phenylborate linker via glucose monomer 1,2-diol groups (Fig. 21). Consequently,

dextran closes the surface of pores, preventing drug release. CPT release mechanism

is based on the competition between soluble glucose and dextran for binding

Fig. 21. Schematic procedure for preparation of glucose-responsive hybrid nanoparticles

with iron oxide cores and dextran-gated mesoporous silica shell. The design involves the
synthesis of magnetic mesoporous silica nanoparticles (MMS) functionalized with
phenylboronic acid and folate. Then, CPT was loaded inside the pores of MMS, the
outside of pores was closed by dextran via binding with phenylboronic acid. Dextran-
gated pores were opened for drug release in the presence of glucose that competes
binding with phenylboronic acid. (Reprinted with permission from ref. 199. Copyright ©
2014 American Chemical Society)

55
phenylboronic acid. Glucose has one 1,2-cis diol group that binds strongly to

phenylborate linker, removing the larger size dextran from pore surface. In vitro results

over HeLa human cervix carcinoma cell line showed a significant reduction of cell viability

in glucose containing RPMI cell culture medium, but no effect in the absence of glucose,

showing that drug loaded particle offers glucose concentration-dependent cell viability.

To end this section, we can summarize that stimuli-responsive systems are able to

deliver CPT to tumor tissue and cells with minimum (or meaningless) premature release,

imposing an efficient drug discharge inside cells under a specific stimulus. This is

particularly important in the case of very cytotoxic molecules like CPT, as these DDSs

increase anti-tumor activity and minimize side effects. It must be taken into account that

for most DDSs, less than ~5% of the injected dose reaches the tumor site [161]. In this

context, the design of nanocarriers sensitive to exogenous or endogenous stimuli

represent a safe alternative to regular chemotherapy with CPT structural analogues. The

main concern with most of these systems, for translation from the bench to the bedside,

is related to their chemical complexity, as most of them are hybrid materials with organic

and inorganic components, and several-step synthesis protocols. This hampers not only

the scaling-up of their synthesis, but also their degradability and biocompatibility.

Moreover, the ability of these models to be sensitive to discrete variations of pH, redox

potential or enzyme activity is not always straightforward to achieve, whereas the scope

of externally applied stimulus is strongly depends on the penetration depth ability.

Nevertheless, some of these systems are on the way towards achieving clinical

application, as it will be shown below.

5. Clinical testing

One of the most disputable arguments again nanomedicines is that, despite the strong

publication statistics over the last decade, very few drugs have reached the commercial

market yet [79]. Although it is accepted that nanocarriers improve solubility, efficacy and

56
biodistribution, while decreasing adverse side effects of very cytotoxic drugs as CPT,

strict regulations concerning their use in human beings have hampered, so far, a quicker

translation of these therapeutic platforms into the clinical stage. At this point, it must be

taken into account that nanocarriers are evaluated for each of their components within

the formulation, and aspects like colloid stability in biological fluids, low immunogenicity

and long-term toxicity may not always be assessed. Moreover, the necessary refining of

the delivery process performance is yet to be achieved [200]. In this context, only a few

organic polymers have been approved by the Food and Drug Administration (FDA) as

CPT delivery systems for cancer therapy. Below we describe briefly some of the most

representative examples as well as their current position at the pipeline. All these cases

have been compiled in Table 2. We accept that some of these DDSs might not form solid

nanoparticles in real in vivo conditions [201], but in all cases, the methods followed for

material preparation, drug administration, biodistribution profile study and therapeutic

validation are fully comparable.

CRLX101 (formerly IT-101) is a pharmaceutical polymeric nanoparticle developed at

Calando Pharmaceuticals, Inc. composed of a cyclodextrin-PEG copolymer covalently

linked to CPT, and was designed to enhance drug solubility and biodistribution [117].

The antitumor drug was released by hydrolysis of the ester bond between the

cyclodextrin and CPT hydroxyl group at C20 position. With regards to irinotecan, this

compound showed improved cytotoxicity against MDA-MB-231 human breast cancer

cells, Panc-1 human pancreatic cells and HT29 human colorectal adenocarcinoma cell

line [117,202], entering Phase Ia trial for the treatment of solid tumors over 10 patients

in 2006. Also, a combined Phase Ib/IIa trial over 62 patients with advanced solid

malignancies demonstrated encouraging safety, pharmacokinetic, and efficacy [203].

This preparation was tolerated with no evident side effects. Consequently, a Phase II

trial was started in 2008 for therapy of 29 ovarian cancer patients and, later on, in 2011,

a Phase II trial was launched over 157 patients with non-small cell lung cancer, in order

57
to assess the activity and safety of CRLX101. In addition, comparative preclinical studies

suggested that CRLX101 behavior in animal models could be the same than in humans

[204]. Actually, we should consider that, although nanomedicine accumulation in solid

tumors by EPR effect is quite rationalized for animal models of human disease, these

models poorly represent human tumors. Nevertheless, in a very recent study over 9

gastric tumor patients consisting of collecting tumor and nonneoplastic tissue biopsies

24-48 h after CRLX101 administration, evidence indicated an intact deposition of

nanoparticles in tumor tissue in five of these 9 patients, whereas no CRLX101 trace was

found in nonneoplastic tissue. Moreover, in all cases, sufficient CPT concentration

reached the tumor to cause down-regulation of topoisomerase I and carbonic anhydrase

IX [205].

CT-2106 is a CPT-poly-L-glutamate conjugate developed to improve the solubility and

prevent CPT lactone ring cleavage. In this supramolecular system, CPT binds the

glutamate polymer through esterification at 20-OH. In preclinical models, CT-2106

showed antitumor activity against multiple tumor cell lines, such as B16 murine

melanoma cells and H322, H460 and H1299 human lung cancer cells, and retained

substantial anti-tumor activity in syngeneic (over C57BL/6 mice) and xenogeneic (over

female nude mice) tumor models [206]. CT-2106 Phase I trial was started by Cell

Therapeutics, Inc. in 2003. A dose escalation study was carried out over 24 patients with

different advanced solid tumors [207]. The only serious related toxicity was grade 4

hypersensitivity in a patient with a history of hypersensitivity to irinotecan. Furthermore,

Cell Therapeutics, Inc. extended this Phase I trial in 2006. For this sake, 26 patients with

advanced solid tumors (50% melanoma), were intravenously weekly administered for

three consecutive weeks during a 28-day cycle [208]. Dose limiting toxicities were

thrombocytopenia and fatigue. The pharmacokinetic profile of conjugated CPT showed

rapid urinary excretion, and similar terminal half-life than free drug. 25 patients were

assessable for response, but no objective responses (complete or partial) were noted.

58
Only 3 patients had stable disease, one breast cancer patient, one histiocytoma and one

melanoma, whereas the rest exhibited progression of the disease. Overall, this trial

provided a strong indication that CT-2106 may have the ability to substantially improve

the toxicity profile of standard CPT.

XMT-1001 is a water soluble macromolecular conjugate (Mw~70 kDa) synthesized by

tethering CPT by hydroxyl group at C20 position to a hydrophilic, biodegradable

polyacetal polymer, poly(1-hydroxymethylethylene hydroxymethylformal), also called

PHF or Fleximer® [209]. This compound was designed to improve efficacy and toxicity

with regards to CPT, and showed higher antitumor efficacy compared with irinotecan

against HT-29 human colon carcinoma xenografts [210]. CPT discharge involves a dual-

step release mechanism: first, XMT-1001 follows an intramolecular transacylation

releasing two CPT prodrugs, camptothecin-20-O-(N-succinimidoglycinate) (CPT-SI) and

camptothecin-20-O-(N-succinami-doyl-glycinate) (CPT-SA) and, then, these prodrugs

hydrolyze to yield CPT. A Phase I study with XMT-1001 was lauched in 2007 by Mersana

Therapeutics, Inc. to determine the maximum tolerable dose (MTD), and to assess the

safety and pharmacokinetics of XMT-1001 and its release products. Then, a total of 24

patients with small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC)

received XMT-1001 by intravenous infusion every 21 days [211]. No drug related serious

adverse events were observed during treatment, whereas several patients showed

promising evidence of clinical activity, including tumor shrinkage and stable disease for

several weeks. Also, preliminary pharmacokinetics findings confirm the formation of

release products (CPT-SI, CPT-SA, and CPT) in plasma. Subsequently, a Phase Ib

extension study of XMT-1001 was initiated in 2011 over 11 patients (10 with NSCLC and

1 with gastric cancer), aiming at fully exploring the broad anti-tumor potential of this

formulation, but no further results have been reported yet.

59
Table 2. Nanomedicines of CPT under clinical investigation.a
Clinical Number of
Compound Formulation Pathology Dosis regimen MTDb Toxicity Ref.
trial patients
Advanced Gastric,
60 minute IV infusions on
Gastroesophageal, or
CRLX-101 Cyclodextrin-PEG copolymer Phase Ia 10 days 1, 8, and 15 of a 28 30 mg/m2/month Pancytopenia [202]
Esophageal Squamous or
day cycle
Adenocarcinoma
60 min IV infusion for 3 Myelosuppression,
Phase 15 mg/m2 IV every
62 Solid malignances consecutive weeks every grade 3/4 neutropenia [203]
Ib/IIa 2 wks
28 days and fatigue.
Ovarian Cancer IV infusion once every 14 G3 vasovagal reaction
15 mg/m2 IV every
Phase II 29 Fallopian Tube Cancer days. Each cycle is 28 G3 pneumonia G3 [117]
2 wks
Primary Peritoneal Cancer days pulmonary embolism
15mg/m2 IV every other 15mg/m2 IV every Fatigue/asthenia,
Phase II 157 Non-small cell lung cancer [117]
week 2 wks dypsnea
Neutropenia and
thrombocytopenia,
Poly(L-glutamic acid) polymer 10 min IV infusion every 3 75 mg CPT-
CT-2106 Phase I 24 Adult solid tumors febrile neutropenia, [207]
conjugate wks 2106/m2
grade 2 hematuria,
stomatitis
Grade 3 fatigue and
Advanced solid tumors (50% 10 min IV infusion weekly 25 mg CPT- grade 3 and 4
Phase I 26 [208]
melanoma) x 3 every 4 wks 2106/m2/week thrombocytopenia,
grade 2 hematuria
Polyacetal poly(1-
Small and non-small cell 1-20.5 mg CPT eq/m2 IV 113 mg CPT Myelosuppresion,
XMT-1001 hydroxymethylethylene Phase I 24 [211]
lung cancer infusión once every 3 wks (eq)/m2 diarrhea
hydroxymethylformal)
10 with non-small cell lung
IV infusion once every 3 initially 113 mg
Phase Ib 11 cancer and 1 with gastric --- [209]
wks CPT (eq)/m2
cancer
7000 mg/m2 Myelosuppression,
Solid malignances (15 with 1 h IV infusion once every
EZ-246 Pegylated CPT conjugate Phase I 37 (1.67% CPT bladder toxicity at [213]
colorectal cancer) 3 wks
weight load) 4800 mg/m2
Advanced and metastatic
Neutropenia,
adenocarcinomas of the 1 h IV infusion once every
Phase II 35 3240 mg/m2 thrombocytopenia, [214]
stomach gastroesophageal 3 wks
anaemia
junction
Metastatic, refractory solid 100 mg Myelosuppression,
N-(hydroxypropyl)methacrylamide 30 min IV infusion once
MAG-CPT Phase I 23 tumors (6 with colorectal CPT/equiv./m2 grade 2 [218]
polymer every week
cancer) every 4 wks dysuria/hematuria
a Additionalinformation may be found at www.clinicaltrials.gov.
b MTD: Maximun tolerated dose
Conversely, EZ-246 (also known as pegamotecan) is a covalent conjugate of CPT with

poly(ethylene glycol) (Mw~40 kDa), developed by Enzon Pharmaceuticals. In preclinical

in vivo studies, this compound showed higher activity than irinotecan and topotecan

against human tumor xenografts (including colon, lung, breast and pancreatic origin)

[212]. Then, in a Phase I trial of this formulation, 37 patients with different solid

malignancies (15 with colorectal cancer) were administered by intravenous infusion for

1 h every 3 weeks. Antineoplastic activity was observed in 2 patients, but significant

toxicity was associated to the treatment, mostly neutropenia and genitourinary toxicity

[213]. Actually, free CPT accumulated slowly in plasma, with a dose proportional

pharmacokinetics, which forced to reduce the dose limiting toxicity in 6 patients.

Subsequently, a Phase II trial was started at 2004 over 35 patients with advanced and

metastatic adenocarcinomas of the stomach and gastroesophageal junction [214-215].

Partial antitumor response was observed in 5 subjects, with a median time to progression

of about 12 weeks, and median overall survival of 38 weeks. However, severe toxicity,

including neutropenia, thrombocytopenia, fatigue, nausea, vomiting and anorexia,

appeared again in some patients, showing the need of further studies, maybe combined

with other active agents.

Finally, MAG-CPT (also known as PNU 166148 and mureletecan) comprises CPT linked

to a water-soluble polymeric backbone methacryloylglycynamide (Mw~18 kDa),

developed by Pharmacia. Therefore, CPT derivatives were synthetized by conjugation

of a N-(2-hydroxypropyl)methacrylamide copolymer precursor with modified CPT at the

C20 α-hydroxy with glycine [216]. These conjugates were designed primarily to improve

the clinical delivery of CPT, and utilized a hydrolytically labile spacer able to liberate drug

intratumourally in a pH and enzyme-dependant manner. Preclinical studies of MAG-CPT

showed that this compound was active in colon, stomach, pancreas, ovary, breast, lung,

and melanoma xenograft models [217]. Then, a Phase I study over 23 patients with

metastatic, refractory solid tumors (6 with colorectal cancer) was carried out in 1999
[218]. Unfortunately, no objective antitumor responses were seen, and severe drug-

related toxicity effects were reported, as myelosuppression, neutropenic sepsis and

diarrhea. Also, one patient died after the first treatment cycle at the maximum dose.

Based on the results of the Phase I studies and the intratumoural kinetic study,

Pharmacia made a strategic decision to discontinue further clinical development of MAG-

CPT [219].

6. Conclusion and future directions

CPT is one of the most active molecules for cancer treatment, but its particular limitations

as poor solubility, lack of stability of the lactone ring and strong toxicity, have encouraged

the development of different delivery forms in order to achieve a safe approach. Here, it

is a fact that current cancer treatment cannot be tackled as the administration of a single

therapeutic molecule but, instead, the new target is personalized medicine, which selects

the appropriate drug combination and dosing schedules for individual patients.

In this context, despite the fact that irinotecan and topotecan present improved

pharmacokinetic parameters, and that they are the only commercial CPT derivatives

approved by the FDA, therapies merely relying on single, small molecules seem now out

of step, mostly due to their limited efficacy and unacceptable toxicities. For this reason,

huge emphasis is being placed on developing new DDSs which are able to

simultaneously deliver CPT and other active agents, as well as incorporating imaging

moieties that may ease the diagnosis process. Most specifically, future seems good for

those systems which are able to selectively discharge the drug at the target cells under

an specific stimulus, with no previous release. Stimuli-responsive preparations provide

a dramatically better spatial and temporal control over drug release, which is crucial for

delivery of the very cytotoxic CPT.

62
Although no CPT nanomedicine has achieved commercial form, there are several

organic polymer compositions currently under different phases of clinical trials. In all

cases, there is a significant increase of the MTD with regards to CPT structural

derivatives. Among them, cyclodextrin conjugates as CRLX101 seem to be the best

positioned to achieve regular clinical use, according to the promising efficacy and

tolerability results, as well as plasma half-life values obtained in Phase II trials. However,

most of these systems show limited functionalization ability to bind covalently the drug,

which limits the loading capacity, as well as the possibility to combine CPT with other

therapeutic molecules and/or imaging agents. In this sense, next generation of CPT

delivery vehicles should merge properties of organic and inorganic materials. Whilst

organic components will provide good stability in physiological fluids, low toxicity and

immunogenicity, and minimized reactions with serum proteins, inorganic materials

(which alone could present strong limitations for biological use, due to possible

compositional toxicity and severe interaction with serum proteins), may offer almost

unlimited external surface area to bind different active molecules and stimuli-sensitive

linkers and devices. Obviously, from the regulatory point of view it is much more

challenging to develop hybrid nanocarriers, which require the evaluation of every single

component and, possibly, their clinical translation will not be as smooth as that of organic

polymers, but the potential of these systems points out to solving the strong interpatient

variability that currently hampers the widespread use of CPT nanopreparations. In this

review, we have tried to reflect some of the most interesting candidates to achieve the

clinics soon.

Acknowledgements

Financial support of the Spanish Ministry of Economy and Competitiveness (projects

MAT2012-39290-C02-02 and SEV-2012-0267) is gratefully acknowledged. Dr. E.M.

63
Rivero thanks the Cursol Foundation for a post-doctoral scholarship. Prof. Eduardo

Fernández and Dr. Ibane Abasolo are acknowledged for the useful discussion.

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