International Journal of Pharmaceutical Sciences and Drug Research 2019; 11(4): 141-146

ISSN: 0975-248X
RESEARCH ARTICLE CODEN (USA): IJPSPP

Formulation and Evaluation of Copper Nanoparticles Loaded

Microsponges
S. Kothai, R. Umamaheswari*
Ethiraj College for Women, Chennai-600 008, Tamil Nadu, India
Copyright © 2019 S. Kothai et al. This is an open access article distributed under the terms of the Creative Commons Attribution-
NonCommercial-ShareAlike 4.0 International License which allows others to remix, tweak, and build upon the work non-commercially, as long as
the author is credited and the new creations are licensed under the identical terms.

ABSTRACT
Microsponges become imperative in the field of targeted drug delivery and in other biomedical applications. There
was a clamant need for designing microsponges incorporating with green synthesised metal nanoparticles rather
than the chemical drug in order to reduce the side effects of the drug and thus increasing the effectiveness of nature
of the whole material. It provokes us to design this novel approach of loading copper nanoparticles into the
microsponges. Here in this work, microsponges based on ethyl cellulose and polyvinyl alcohol were synthesised by
Quasi-Emulsion Solvent diffusion method in which copper nanoparticles procured from Hibiscus rosa-sinensis leaf
extract was incorporated. The Loaded microsponges were characterised by High Resolution Scanning Electron
Microscopy (HR-SEM) and Particle size distribution Analyzer (PSA). The Drug content and Entrapment Efficiency of
the microsponges were found out. The antimicrobial and antioxidant activity of the loaded microsponges were
evaluated.

Keywords: Copper nanoparticles, microsponges, HRSEM, PSA, antimicrobial, antioxidant.

DOI: 10.25004/IJPSDR.2019.110407 Int. J. Pharm. Sci. Drug Res. 2019; 11(4): 141-146

*Corresponding author: Mrs. R. Umamaheswari

Address: Department of Chemistry, Ethiraj College for Women, Chennai-600 008, Tamil Nadu, India
E-mail : [email protected]
Relevant conflicts of interest/financial disclosures: The authors declare that the research was conducted in the absence of any commercial or
financial relationships that could be construed as a potential conflict of interest.
Received: 26 June, 2019; Revised: 25 July, 2019; Accepted: 26 July, 2019; Published: 30 July, 2019

INTRODUCTION scenario, the development of microsponge loaded with

Microsponges are polymeric delivery systems, specific drug has been emphasised due to their
possessed of porous polymeric microspheres that can controlled release of the drug. Since the microsponges
entrap active ingredients. These are tiny sponge like prepared from synthetic polymers, it will protect the
spherical particles that consists of myriad of entrapped drug from any kind of degradation. These
interconnecting voids with large porous surface. kinds of encapsulated drugs within microsponge
Usually the size of the microsponges range from 5 to system can significantly reduce the irritation, side
300µm. [1-2] Metal nanoparticles such as gold, silver and effects of the drug without decreasing its efficiency. [7-8]
copper are reported as highly toxic to micro-organisms. The current work involves the formulation and
[3-4] In recent years, it has been extensively used for the evaluation of copper nanoparticles loaded
production of medical products like wound dressing microsponges and its biomedical applications. Here,
because of its strong cytotoxicity. [4-6] In current microsponges were synthesised by Quasi-Emulsion
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Solvent diffusion method using different proportions of After incubation, the precipitate got settled down that
ethyl cellulose and polyvinyl alcohol. Later, the green was confirmed by the colour change from green to
synthesised copper nanoparticles from the leaf extract black. This indicates the formation of Copper
of Hibiscus rosa-sinensis were incorporated into the nanoparticles that was purified by repeated
microsponges. The formulated and loaded centrifugation at 6000 rpm for 10 min to remove
microsponges were characterised by SEM and PSA. The unwanted materials. The synthesized CuNps were
Drug Content and Entrapment Efficiency of the loaded lyophilized and stored at 25°C for further use. [9]
microsponges were studied. The antimicrobial and Synthesis of copper nanoparticles loaded
antioxidant activity of the copper nanoparticles loaded Microsponges
microsponges were evaluated. Copper nanoparticles loaded Microsponges were
Hence, in the present work an attempt was made for formulated by Quasi-Emulsion Solvent Diffusion
the first time by incorporating copper nanoparticles in method. Five batches of microsponges (NS 0 – NS4CuB)
the microsponges rather than any chemical drug. These with varying proportions of Ethyl Cellulose (EC) and
metal nanoparticles loaded microsponges will minimise Polyvinyl alcohol (PVA) were taken. The Dispersed
the toxicity of the drug intake, prolong the Phase consists of Copper Nanoparticles (B -CuNps) and
pharmacological effect and thus improve the overall required amount of EC dissolved in 20 mL of
effect of the microsponges. The copper nanoparticles Dichloromethane (DCM). It was slowly added to PVA
loaded microsponges will show enhanced activity in 150 mL of aqueous continuous phase. Then it was
towards biomedical applications than the copper stirred at 1000 rpm under magnetic stirrer for 3 hours.
nanoparticles alone. In future, this study would lead to The microsponges formed were filtered and dried in
a new scenario of introducing copper nanoparticles oven at 40–50ºC for 24 hours. Then the dried
loaded microsponges for a smarter application. microsponges were stored in vacuum dessicator to
remove the residual solvent. The composition of the
microsponge formulation was tabulated in Table 1. The
Figure 2 indicates the schematic representation of
microsponge formation. The prepared microsponges
were characterized based upon the entrapment
efficiency and particle size. [8]

Table 1: Formulation of Nanosponges with (B) CuNps

Copper
Sample PVA EC DCM H2O
Nanoparticles (B)
Code g g mL mL
mg
NS0 - 2 2 20 150
NS1CuB 10 2 2 20 150
NS2CuB 10 2 3 20 150
Fig. 1: Graphical Abstract of the current work NS3CuB 10 3 2 20 150
NS4CuB 10 3 3 20 150

MATERIALS AND METHODS

Materials
Ethyl Cellulose (EC), Polyvinyl alcohol (PVA),
Dichloromethane (DCM) of reagent grade were kindly
purchased and used without purification. Copper
nanoparticles were green synthesized from the leaf
extract of Hibiscus rosa-sinensis. Double Distilled water
was used throughout the study.
Green synthesis of copper nanoparticles
The Copper nanoparticles (B) were synthesized from
the leaf extract of Hibiscus rosa-sinensis. The fresh leaves
were collected and washed with distilled water to Fig. 2: The schematic representation of microsponges formation.
remove dust and impurities and shade-dried for 3-4
days at room temperature. About 100 g of dried and Characterisation of Copper nanoparticles loaded
minced leaves were weighed and transferred to beaker Microsponges
containing 100 mL distilled water. It was then boiled at Microscopic studies
60°C for 10-15 min. First, the prepared solution was The morphology of the loaded microsponges and
filtered Whatmann no.1 Filter paper to get a clear unloaded microsponges was studied by using High
solution. This filterate was known as Hibiscus rosa- Resolution Scanning Electron Microscopy (HRSEM).
sinensis leaf extract. 50 mL of this extract was added to Here we have used VEGA3 TESCAN instrument for
50 mL of 0.05M CuSO4, kept for incubation for 3 days. our characterization work. A thin film of the sample

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was made by placing a pinch of the sample on a carbon added and 10μL of fungal suspension was added on
coated grid and then the film on the SEM grid was each well. Each plate was wrapped loosely with cling
made to dry under mercury lamp for 5 minutes. film to ensure that bacteria did not become dehydrated.
Particle size determination The plate was incubated at 37°C for 18–24 hours. The
The particle size of the copper nanoparticles loaded colour change was then assessed visually. Any colour
Microsponges was determined by using Particle Size changes from purple to pink or colourless were
Distribution Analyzer. Here the instrument used was recorded as positive. The lowest concentration at which
HORIBA Laser Scattering Particle Size Distribution colour change occurred was taken as the MIC value. [9]
Analyzer LA-950. Antioxidant study
Percentage Yield Determination of scavenging activity by DPPH assay
The percentage yield of copper nanoparticles loaded The percentage of antioxidant activity (AA %) of each
microsponges of various batches were calculated using substance was assessed by DPPH free radical
the weight of final product after drying with respect to scavenging assay. Different concentrations of sample
the initial total weight of drug and polymer used for were added to all the tubes except blank. Then 3 mL of
the preparations. [8] ethanol and 0.3 mL of 0.5 mM DPPH radical solution in
Drug Content and Entrapment Efficiency ethanol was added. The control solution was prepared
About 10 mg of microsponge from all batches were by mixing ethanol (3.5 mL) and DPPH radical solution
accurately weighed and dissolved in methanol in 50 (0.3 mL). Absorbance was read at 517 nm after 30 min
mL standard flask and then made up to the volume of of reaction. [9] The scavenging activity percentage (AA
phosphate buffer pH 7.4. After appropriate dilution, %) was calculated using the below formula
the amount of drug was detected by a UV % Antioxidant activity = {(absorbance at blank) –
Spectrophotometric method at 650 nm using blank (absorbance at test) / (absorbance at blank)} × 100
microsponges treated in the same manner. [8] The
Entrapment Efficiency was calculated according to the RESULTS AND DISCUSSION
following equation: Microscopic studies
Entrapment Efficiency (%) = [Actual drug content in From SEM studies, it was found that the samples had
microparticles/Theoretical drug content] × 100 porous and almost spherical sponge in nature. The
Preparation of Standard Calibration Curve pores were induced by the diffusion of the solvent. [8]
Preparation of Phosphate Buffer pH 7.4 The SEM image of CuNps(B) were spherical and
Phosphate Buffer was prepared and pH was found to agglomerated to form clusters (Fig. 3). [9] The unloaded
be 7.4 using digital pH meter. [8] Microsponges shows shiny smooth surface morphology
Determination of λmax of copper nanoparticles (Fig. 4). The Loaded microsponge shows porous
The absorption maxima for copper nanoparticles (B) smooth surface and spherical (Fig. 5). SEM results
were found to be 650 nm. [9] revealed that surface morphology has been shown to be
Standard calibration curve of Copper nanoparticles beneficial for topical application for future studies.
(B) Particle size
The absorbance of copper nanoparticle standard The Particle size analysis of loaded and unloaded
solutions having a concentration range of 100- microsponges (Fig. 6) revealed that the particle size
500µg/mL in phosphate buffer pH 7.4 was plotted. The ranges from 65µm to 93µm. NS4CuB was selected for the
curve was found to be linear at λmax 650 nm. The further study in terms of lower particle size (65µm).
calculation of the drug content and Entrapment The smaller particle size shows better entrapment
efficiency were based on this calibration curve. [8] efficiency in future.
In- vitro antimicrobial study
Determination of Minimum inhibitory concentration
(MIC) using Resazurin Microtitre Assay
Preparation of resazurin solution
The resazurin solution was prepared by dissolving 270
mg in 40 mL of sterile distilled water. A vortex mixer
was used to ensure that it was a well-dissolved and
homogenous solution.
Test was carried out in a 96 well Plates under aseptic
conditions. A sterile 96 well plate was labelled. A
volume of 100μL of sample was pipetted into the first
well of the plate. To all other wells 50μL of nutrient
broth was added and serially diluted it. To each well
10μL of resazurin indicator solution was added. 10μL
of bacterial suspension was added to each well.
Similarly, the same set up was performed for antifungal
Fig. 3: SEM photomicrograph image of Copper Nanoparticles (B)
activity in which 50μL of potato dextrose broth was
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Antimicrobial activity by Resazurin microtitre assay

The synthesised NS4CuB microsponge formulation was
selected for the biomedical applications due to its least
particle size and better entrapment efficiency. The
antimicrobial activity was done by Resazurin Microtitre
assay (Table 3). It shows good antibacterial activity
towards E. coli and B. subtilis whose MIC values are
125µg/mL and 31.2µg/mL respectively. From the
results, it shows more active towards B. subtilis. The
MIC values of copper nanoparticles loaded
microsponge NS4CuB is almost equal to that of the
value of CuNps(B). [9] Similarly, NS4CuB shows an
excellent antifungal activity towards C. albicans whose
MIC value is 62.5µg/mL whereas the MIC value of
Fig. 4: SEM photomicrograph image of unloaded microsponge NS 0 CuNps(B) was found to be 250µg/mL. [9] Hence, it is
proven that the antifungal activity nature of copper
nanoparticles loaded microsponge is enhanced.
(Antibacterial activity – STD- Streptomycin)
(Antifungal activity- STD- Amphotericin B)
Antioxidant activity by DPPH assay
The copper nanoparticles loaded microsponge
formulation NS4CuB has an antioxidant potential of
59.5% (Table 4). The percentage scavenging activity of
copper nanoparticles loaded microsponge is slightly
lower than the value of standard BHT (Fig. 10). The
CuNps(B) showed 21.7% of scavenging activity. [9]
From the results, it shows that the antioxidant activity
increases in the copper nanoparticles loaded
microsponge formulation (NS4CuB). This indicates the
successful encapsulation of drug (CuNPs) within the
microsponge. Therefore, the copper nanoparticles
Fig. 5: SEM photomicrograph image of loaded microsponge NS4CuB
loaded microsponge enhanced the activity of CuNps. It
Particle size (µm) reveals the porous nature of the outer surface of the
sponge offers control on the release of drug.
100

80
particle size (µm)

60

40

20
Fig. 7: Production yield of loaded and unloaded Microsponges

0
NS0 NS1CuB NS2CuB NS3CuB NS4CuB
Fig.Fig 6. Particle
6: Particle size
size of of loadedand
loaded andunloaded
unloadedMicrosponges
Microsponges

Production yield
The production yield of all the microsponges were
calculated and shown in the Fig. 7.
Drug Content and Entrapment Efficiency
The Drug content and Entrapment Efficiency were
calculated and displayed in the table 2.
Standard Calibration Curve
The standard calibration curve for copper nanoparticles
(B) in phosphate buffer pH 7.4 at 650 nm was shown in Fig. 8: Standard calibration curve of CuNps (B)
Fig. 8.

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Table 2: Drug Content and Entrapment Efficiency % of Microsponge formulations

Sample Code Absorbance Concentration Drug Content % Theoretical Drug Content % in 10 mg Entrapment Efficiency %
NS1CuB 0.121 403.33 4.0333 ± 0.0183 0.0497 81.07 ± 0.3438
NS2CuB 0.113 376.67 3.7667 ± 0.2269 0.0332 113.37 ± 6.8016
NS3CuB 0.149 496.67 4.9667 ± 0.1836 0.0497 99.83 ± 3.6701
NS4CuB 0.135 450 4.50 ± 0.0839 0.0332 135.46 ± 2.4837

Table 3: Antimicrobial activity of Copper nanoparticles(B) loaded Microsponge formulation NS 4CuB

S. Microorganisms/sample Growth of inhibition (µg/mL)
No NS4CuB 1000 500 250 125 62.5 31.2 15.6 7.8 STD 10 Sterile water Culture
1 Escherichia coli - - - - + + + + - + +
2 Bacillus subtilis - - - - - - + + - + +
3 Candida albicans - - - - - + + + - + +

and entrapment efficiency were determined. The

NS4CuB with least particle size showed better
entrapment efficiency of 135%. The antibacterial
activity of copper nanoparticles loaded microsponge
formulation NS4CuB shows good activity on B. subtilis.
The MIC values of CuNps loaded microsponge is
equivalent to that of the drug (CuNps). Similarly, the
antifungal activity of NS4CuB towards C. albicans is
increased when compared to that of CuNps. The
antioxidant activity of NS4CuB showed an enhanced
activity of 59.5% to that of the CuNps (21.7%).
In this work, we have made an attempt to incorporate
Fig. 9: Antimicrobial activity representing MIC value of NS4CuB copper nanoparticles in microsponge for the first time.
We have succeeded in our venture by encapsulating
CuNps in the microsponge formulation, thereby
enhancing the activity of the copper nanoparticles. The
smooth and porous nature of the formulation offers
good control on release of the drug and hence it can be
used in topical application in future.

ACKNOWLEDGEMENT
We profusely thank Dr. Sundar Mayavan, Scientist,
CSIR-CECRI, Karaikudi for helping us in HRSEM of
our work.
Fig .10. Antioxidant activity of NS4CuB by DPPH assay.
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HOW TO CITE THIS ARTICLE: Kothai S, Umamaheswari R. Formulation and Evaluation of Copper
Nanoparticles Loaded Microsponges. Int. J. Pharm. Sci. Drug Res. 2019; 11(4): 141-146. DOI:
10.25004/IJPSDR.2019.110407

Int. J. Pharm. Sci. Drug Res. July-August, 2019, Vol 11, Issue 4 (141-146) 146