55

Ciência eNatura, Santa Maria, v. 37 Part 1 2015, p. 55−62

ISSN impressa: 0100-8307 ISSN on-line: 2179-460X

Preparation and In vitro Evaluation of Isoniazid-Containing Dex-

HEMA-Co-PNIPAAm Nanogels

Maryam Jafari 1Babak Kaffashi2

1PhD Student, Department of Chemical Engineering, University of Tehran, Tehran, Iran

2 Assosiated Professor, Department of Chemical Engineering, University of Tehran, Tehran, Iran (Email:
[email protected])

Abstract

In this work, Dex-HEMA-Co-PNIPAAm nanogels containing Isoniazid antibiotic were made. Characteristic features of nanogels were
studied by Fourier transform infrared spectroscopy (FTIR), dynamic light scattering (DLS) and scanning electron microscopy (SEM). Drug
loading capacity and entrapment efficiency were determined. In vitro drug release amount was estimated at room and body tempe rature.
Biocompatibility of gels was investigated through cytotoxicity assay. Finally antimicrobial properties of synthesized gels were studied. It was
shown from the experimental data that the nanogels size after drug loading increased about 1-2%. %Isoniazid loading and %entrapment
efficiency were in the range of 15-22% and 37-48% respectively. After 10 days of degradation ca. 80% at 25ºC and ca. 90% at 37ºC of the
nanogel structures were destructed. No significant toxic product produced while degradation and all nanogels depicted good
biocompatibility. No antimicrobial features observed through the test condition against gram negative E Coli.

Keywords: nanogel, Isoniazid, Dex-HEMA-PNIPAAm, MTT, Antibacterial.

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and branches begin from α-1, 3 links. Dextran

1 Introduction hydrolyze or dextranase degrade the dextran by
endohydrolysis of α-1, 6 glycosidic linkages.

I
n recent years, with the development of
pharmacy many efforts have been done to
improve the drugs performance and reduce Hydrogels, 3D hydrophilic networks, may be
the drugs side effects. Due to low drug building even by physical or chemical cross
absorption, fast metabolism and rapid links. High amount of water can be absorbed by
elimination of drug from body, drug gels and duo to this feature gels are like body
concentration dose not stay steady (Haddadi- tissue. Hence, gels show good biocompatibility
Asl, 2011). To solve these types of problems when using as drug carriers. Stimuli sensitive
introducing new kinds of drug delivery systems hydrogels can control the time of drug release by
using drug carriers seems necessary. phase changes and transient when facing stimuli.
Stimuli can be physical like temperature, light or
electrical and magnetic fields, chemical like pH
Drug delivery includes any formulation and
and finally biological (Franssen, 1999, Dijk-
method capable of drugs molecules convey in to
Wolthuis, 1997, Huang, 2004, Lui, 2011).
the body. Such systems are prepared in different
kinds, like micro/nano particles, micro/nano
capsules, gels and hydrogels, liposomes, micelles Temperature or thermo sensitive gels are among
and dendrimers (Patel Kundan, 2012, .Oreilly, the most investigated drug delivery systems, in
2013. Hang, 2010., Mohammadpour, 2012., which swelling/deswelling occur as the
Michelle, 2013). In addition to drug environment temperature changes. In this way
transportation, drug delivery will enhance drug drug will release in an on/off manner (Lowe,
release, distribution and bioavailability. 2005, Georgiou, 2011).

Lots of materials can be used as drug carriers Poly N-isopropylacrylamide (PNIPAAm) and its
such as metals, ceramics, biomaterials, copolymers are widely used in thermo sensitive
composites and polymers. Among these, in carrier design. This polymer shows negative
accordance with cheapness, light weight, high behavior, which means that they show lower
chemical resistance and different physical, critical solution temperature. In the simplest way
chemical and mechanical properties polymers at low temperature the drug will be released and
can be suitable and worthy candidate as a on high temperatures the system will be off
pharmaceutics carrier. Polymers can be used in (Perju, 2011, Kurecic, 2012, Huang,2004) .
the forms of synthetic, natural or either a
combination of both types (Gemma, ***. Zhe, We have synthesized and characterized a novel
2013. Franssen, 1999, Huang, 2004). drug delivery system previously (Jafari, 2015).
Our system was consisting of dextran
Biodegradable polymers which mostly have biodegradable natural polymer functionalized by
ester, amide and ether functional group in their adding hydroxyl ethyl Meta Acrylate (HEMA) to
structure can break down to pieces. If they are add hydrolysable group which was then
utilized as drug carrier they can release drugs as copolymerized by PNIPAA to make
they are degrading. As the polymer is degrading thermosensitive nanogels. Swelling and LCST
it converted to the small parts that can be temperature of the gels were determined
excreted from the body or can be used for carefully. In this work Isoniazid as a model drug
microorganism in biological process (Aguilar, was loaded to the nanogels and drug loading
2013, Qiu, 2009, Dijk-Wolthuis, 1997,). parameters were evaluated. Thereafter dug
release, degradation, biocompatibility and
antimicrobial features were studied.
Polysaccharides are a group of natural polymers
that can be digested enzymatically. In Dextran, a
polysaccharide made of glucose molecules, the
linear and main chain consists of α-1, 6
glycosidic linkages between glucose molecules,

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 57

2 Experimental methods 2.3 Characterization of Isoniazid loaded

nanogel
2.1 Materials The structures of Isoniazid loaded nanogels,
from 400-4000 cm-1, were investigated by means
Dextran was obtained from Fluka. N- of Fourier transform infrared (ATR-FTIR,
Isopropylacrylamide (NIPAAm) and 2-Hydroxy- Bruker). Each spectrum was resulted from 32
4’-(2-hydroxyethoxy)-2-methylpropiophenone times scan and no preparation was exerted to the
(Irga cure 2959) and MTT color were purchased nanogels solution prior to study.
from Aldrich. 2-Hydroxyethyl methacrylate, 1, 1- The particles size and size changes due to
Carbonyldiimidazole, 4-(Dimethylamino) drug loading, after suspending the nanogels in
pyridine (DMPA), anhydrous Magnesium distilled water with viscosity of 0.89 CP, were
sulphate (MgSO4), N, N’- evaluated by dynamic light scattering method
Methylenediacrylamide (MBA) and mono and (DLS, Brookhaven Instrument Corporation,
dibasic sodium hydrogen phosphate were Brookhaven, Georgia, US) at 657nm.
available from Merck. Isoniazid, dialysis tube The surface morphology of nanogels and
(12000 Dalton) and L929 cell line were provided Isoniazid loaded nanogels were monitored using
by Iran Pasture institute. Triton X-100 and scanning electron microscopy (SEM, Seron
Dimethyl sulfoxide were obtained from scharlau. Technologies Inc., AIS 2100). Samples were
Hydroquinone monomethyl ether, lecithin and coated by gold before scan to make their surface
HCl were provided from Riddle, Pasargad novin conductive.
and Chem.-Lab, respectively. Tetra hydro- furan
(THF), ethyl acetate and chloroform were Table 1: Composition and ingredients amount in
provided by Dae-Jung, South Korea.
feed

2.2 Preparation of Isoniazid loaded nanogel Sample No. 1 2 3 4 5

Dex-HEMA to NIPAAm
0.5 1 2 2 0.5
monomer ratio
Isoniazid loaded nanogels were prepared Cross linking agent (gr) 0.01 0.02 0.01 0.03 0.03
exactly in the same procedure as nanogels were Photo initiator (gr) 0.01 0.01 0.01 0.01 0.01
synthesized previously. In a few words, freeze Isoniazid (gr) 2 2 2 2 2
dried Dex-HEMA, obtained by coupling the
hydroxyl group of HEMA to dextran as 2.4 Loading capacity and efficiency
described by Dijk- Wolthuis, NIPAAm evaluation
monomer, photo initiator, cross linking agent
and Isoniazid with various amounts as given in To study the nanogel yield (NY), Isoniazid
table 1 were mixed and dissolved in PBS (pH=7). loading capacity (LC) and encapsulation
Afterward, these dispersions were added to the efficiency (EE), Isoniazid loaded nanogel
thin soy lecithin film, which was gained after suspensions were centrifuged at room
vacuum evaporation of lecithin-chloroform temperature for 20 minutes at 10000 rpm. The
solution. Resulting dispersions were mixed precipitate that was consisting of Isoniazid
completely and were left motionless overnight. loaded nanogels were collected for additional
Finally, prepared dispersions were extruded 20 procedure such as liposome lyses and frees
times, through a syringe coupled to 450 nm drying. The supernatants containing free
filters to make nano sized polymerization Isoniazid were gathered and investigated by UV-
reactors. Subsequently to avoid polymerization VIS spectrophotometer at the wavelength of 263
outside the formed liposomes the dispersions nm to find out free Isoniazid concentration.
were diluted and then after, radical The nanogel yield, Isoniazid loading percent
polymerization of samples commenced by 30 and entrapment efficiency of nanogels were
minutes UV irradiation. The end products were computed as follow:
achieved after liposomes washed out using triton
x-100.
𝑛𝑎𝑛𝑜𝑔𝑒𝑙𝑠 𝑤𝑒𝑖𝑔ℎ𝑡 × 100
% 𝑁𝑌 =
𝑝𝑜𝑙𝑦𝑚𝑒𝑟 𝑤𝑒𝑖𝑔ℎ𝑡 + 𝑖𝑛𝑖𝑡𝑖𝑎𝑙 𝑖𝑠𝑜𝑛𝑖𝑎𝑧𝑖𝑑 𝑎𝑚𝑜𝑢𝑛𝑡

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 58

𝜇𝑔
% 𝐿𝐶 ( ) was added to each well and the plate was kept in
𝑚𝑙
(𝑖𝑛𝑖𝑡𝑖𝑎𝑙 𝑖𝑠𝑜𝑛𝑖𝑎𝑧𝑖𝑑 𝑎𝑚𝑜𝑢𝑛𝑡 − 𝑓𝑟𝑒𝑒 𝑖𝑠𝑜𝑛𝑖𝑎𝑧𝑖𝑑 𝑎𝑚𝑜𝑢𝑛𝑡) × 100 37 ºC for 3-4 hr. the medium was removed and
=
𝑛𝑎𝑛𝑜𝑔𝑒𝑙𝑠 𝑤𝑒𝑖𝑔ℎ𝑡 150 µL of DMSO containing formazan salt was
𝜇𝑔
% 𝐸𝐸 ( ) added to each well and read on microplate
𝑚𝑙
(𝑖𝑛𝑖𝑡𝑖𝑎𝑙 𝑖𝑠𝑜𝑛𝑖𝑎𝑧𝑖𝑑 𝑎𝑚𝑜𝑢𝑛𝑡 − 𝑓𝑟𝑒𝑒 𝑖𝑠𝑜𝑛𝑖𝑎𝑧𝑖𝑑 𝑎𝑚𝑜𝑢𝑛𝑡) × 100
= reader. Tests were performed in triplicate. The
𝑖𝑛𝑖𝑡𝑖𝑎𝑙 𝑖𝑠𝑜𝑛𝑖𝑎𝑧𝑖𝑑 𝑎𝑚𝑜𝑢𝑛𝑡
absorbance of samples was observed at 570 nm,
which is sign of cell growth and proliferation.
2.5 In vitro release studies
2.8 Antimicrobial studies
The in vitro drug release from Dex-HEMA-
Co-PNIPAAm nanogels was tested by dialysis The antimicrobial specification of isoniazid
method. Particular mass of lyophilized Isoniazid loaded nanogels were assest against E Coli
loaded gels were weighted and subsequently (ATCC 25912, PTCC 1399), gram negatine
suspended in 3 ml of PBS (pH = 7.4) and poured organism, by help of zone of inhibition test. 1.5*
in individuals dialysis bag. Afterwards each 108 CFU ml-1 of E Coli was prepares and
dialysis bag was placed in separate glass beaker cultivated on LB Agar medium. The suspention
filled with 15 ml of PBS (pH = 7.4), incubated at of 200 mg ml-1 of samples were made in distiled
37 ºC, while was stirred. At different time points, water . disks were soaked in sample suspentions
the absorptions of PBS solution, out of dialysis and then were placed on plated. Plates were
tube, were measure by UV-VIS spectroscopy incubated at 37ºC for 72 hr.
(LKB Biochrom 4050 ultrospec II UV-VIS
spectrophotometer) at the wavelength of 263 nm 3 Results and discussion
to find out the released Isoniazid concentration.

3.1 Isoniazid loaded nanogels

2.6 Hydrolytic degradation studies
characterization
5 mg of dispersed nanogels in PBS buffer Figure 1 illustrat the isoniazid loaded
solution were poured in polyethylene tubes and nanogels spectra. Peaks relating to water (OH~
then the tubes were incubated at 37ºC for 14 1630, H-O-H~3300) are vividly distinguishable.
days. At different time intervals, sample were Peaks belong to Free NH2 (~1220), NH bend
collected and probed by DLS to evaluate the (~1650), pyridine group (~ 1440) of isoniazid
nanogels diameter changes which represent the could been recognized, too.
degradation of the nanogels. The size, size increase of drug loaded
nanogels to empty nanogels and PDI mesured
2.7 Biocompatibility studies by DLS are given in table 2 .
SEM images have demonstrate the
The biocompatibility studies were performed morphology and size of nanogels. The nanogel
by cytotoxicity and cellular viability using MTT were almost spherical and had an smoot
(3-(4, 5-Dimethylthiazol-2-yl)-2, 5- surface, and size range about 400nm which is
Diphenyltetrazolium Bromide) assay. The given in table 2 with more detailes. From the
cytotoxicity of Isoniazid loaded five different camparison of size data catched by both method
series of nanogels were determined facing L929 it is notable that possibly because of freez dried
cells. A 96- well was used in order to seeding particle cogulating to each other bigger size
cells. Each well was then filled with 100 µL of have been gotten.
DMEM medium with 10 % PBS and was
maintained at the temperature of 37 ºC in CO2
atmosphere. One day in the rear of plating,
50,100,200, 500 µg ml-1 of Isoniazid loaded
nanogels were added to the wells. After 24, 48,
72 hr the medium was removed, washed and
then 50 µL of 5 µg ml-1 MTT solution in PBS

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1
2
3
4
5
Figure 2. (top row) Isoniazid free nanogel size,
(bottom row) Isoniazid loaded nanogels size
400 1200 2000 2800 3600 measure by DLS and SEM respectively
Figure 1. Isoniazid loaded nanogels FTIR
spectra 3. 2 Loading capacity, encapsulation
efficiency and nanogels yield
Table 2: Size and size distribution obtained
by DLS and SEM comparison The amounts of Isoniazid which loaded in
nanogels were measured by subtracting the free
Isoniazid value from initial Isoniazid content.
Isoniazid loaded nanogel

Size increase after drug

Size increase after drug

Isoniazid loaded nanogel

Nanogels yield, Isoniazid loading and

loading (%)-SEM
loading (%)-DLS
Size (nm)- DLS

entrapment efficiency were calculating according

Size (nm)- SEM
Sample name

to formulas (1) to (3) are reported in table 3.

PDI

As it is obvious, the less the cross linking

agent was used the more drug loading and
entrapment efficiency percent were resulted.

Table 3: Characteristic of Isoniazid loaded

nanogels
1 339.9 1.46 0.255 424.2 2.38
2 349.8 0.75 0.263 565 2.52 %
Sample % Isoniazid % entrapment
3 354 1.51 0.269 406.6 0.95 nanogels
name loading efficiency
4 329 0.86 0.238 352.5 1.48 yield
5 373.5 1.55 0.268 439.3 1.99 1 73.42 21.77 48.11
2 72.96 18.32 40.31
3 72.92 20.86 45.78
For instance figure 2 represents DLS and
4 72.51 20.90 37.81
SEM images of sample 2 with and without 5 71.85 15.29 38.18
Isoniazid.

3. 3 In vitro Isoniazid release study

while a part of synthesized nanogels

consisted of PNIPAAm, a thermosensitive
polymer, whole nanosized carrier found the
thermoresponding behavior in which the gel
swell and deswell according to temperature
shifts and the net mesh size could change
automatically. Hence the release behavior in
both temperatures limits, below and upper the
LCST temperature, were studied to evaluate the
differences in release mechanism. The results of
Isoniazid release during 10 days from gels at
25ºC and 37ºC is depicted in figure 3.

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 60

As the results show at 37º C and at the

100 25 ͦC
temperature upper LCST which is ca. 32.5º C, in
the physiological conditions nanogels shrink and
the portion of their trapped content were
released suddenly which is observable as a burst 75

% Isoniazid release
release in figure 3. After about 1 hr that all 5
series of freeze dried Isoniazid loaded nanogels
reached the media temperature they deswelled 50 1
and 69.88, 80.72, 88.52, 79.38 and 67.82 % of their
2
content including Isoniazid released
respectively. Then the release regime in all gels 3
25
followed the same way slowly by the
4
degradation mechanism and finally after 10 days
the drug release percent touched the 82.61, 90.38, 5
96.80, 89.73 and 79.95% respectively. The sample 0
2 having the mix combination of the highest ratio 0 24 48 72 96 120144168192216240
of Dex-HEMA to PNIPAAm, having the most Time(hr)
amount of hydrolysable ester bond in its
structure and cross linking agent of 0.01 gr whish
was the less value showed the most amount of 37ºC
release among other samples. Next ranks of 100
Isoniazid release belong to samples 3 and 4
respectively. The effect of Dex-HEMA to
PNIPAAm ratio that results in better 75
% Isoniazid release

degradation were more serious than the cross

linking agent. Last places were occupied by rel
sample 1 and 5 by the previous analysis. 50 ea
At 25ºC that is bellow LCST temperature the se
2
release behavior was different. At this rel
temperature nanogels swelled and the release 25 ea
mechanism was controlled by diffusion. The se
ultimate values for Isoniazid release after 10 3
days were 80.46, 80.08, 79, 77.83 and 77.97 for all 0
the 5 series. As the results represent the values 0 24 48 72 96 120 144 168 192 216 240
are almost similar and no tangible differences Time(hr)
were filled, but even in this case the impact of
cross linking agent can be seen. The more the
Figure. 3. Drug release% from nanogels at 25
cross linking agent observed the less swelling
and 37ºC
and consequently less release attached.
3. 4 Isoniazid loaded nanogels degradation

To study the hydrolytic degradation,

Isoniazid loaded nanogels of different
composition incubated in physiological
condition. At very beginning times an increase in
gels size could be observed which sign of freeze
dried gel swelling was. The swelling was not
considerable because the testes were holding on
upper LCST temperature in which nanogels
were in their shrinking form. Then after the gels
sizes have been decreased slowly within 2

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 61

weeks. Figure 4 shows the percent of particle size time intervals, as the concentration raised, the
decrees as a function of degradation time. cell viability fallen.
As results depict, size decrease due to
hydrolytic degradation occurred most in sample 3. 6 In vitro antimicrobial assay
4, 3, 2, 1 and 5 respectively that can be related to
the quantity of hydrolysable group which is The antimicrobial behavior of nanogels tested
found in Dex- HEMA part of the nanogels. against gram negative E Coli. Unfortunately, not
120 any traces of inhibition zone were detected in all
5 series of samples. According to Gerald Tritz
% Particle size decrease

100
work inhibitory nature of E coli by Isoniazid
80 depends on the initial cell concentration,
Isoniazid concentration, and chemical
60 1
composition of the medium, hence theses
2 parameters could be the origin of losing
40
3 antimicrobial feature of the Isoniazid loaded
20 4 nanogels.
5
0
0 2 4 6 8 10 12 14 4. Conclusion

In this work, Isoniazid loaded biodegradable

Time (Day)
and thermosensitive nanogels were prepared
through UV polymerization method. By
Figure 4. Particle size decrease % due to incorporating PNIPAAm in the nanogels
hydrolytic degradation structure the thermosesitivity characteristic was
given to the gels which affected the degradation
and drug release from the gels in physiological
3. 5 In vitro cytotoxicity assay condition. Hydrolytically degradable
specification also donated to nanogels because of
The cytotoxicity of Isoniazid loaded nanogels Dex-HEMA portion. The degradation product
against L929 cells was evaluated and outcomes were nontoxic but had not any antimicrobial
are represented in figure 5. features. Totally these nanogels seem to be a
good candidate in controlled drug delivery.
100
% survival

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