Sacrificial sulphonated polystyrene template-assisted synthesisof mesoporous hollow core-shell silica nanoparticles fordrug-delivery application

DEEPIKA DODDAMANI and JAGADEESHBABU PONNANETTIYAPPAN*

National Institute of Technology Karnataka, Surathkal, Mangalore 575025, India

*Author for correspondence (dr.jagadeesh@yahoo.co.in)

MS received 7 September 2019; accepted 19 January 2020

Abstract. Spherical mesoporous hollow core-shell silica nanoparticles (HCSNs) of size 200 ± 50 nm with tunable

thickness from 20 to 60 nm are synthesized using a sacrificial sulphonated polystyrene (PS, particle size 160 nm)

template. A facile method is adopted for the sulphonation of PS using sulphuric acid, which enhanced the negative

charge on the surface of PS as confirmed by zeta potential analysis and Fourier transform infrared radiation analysis.

The thickness of the silica shell is tuned by altering the concentration of the silica precursor and is found to increase

due to the use of the sulphonated PS template. N2 adsorption/desorption studies reported the variation of specific

surface area of HCSNs from 644.1 to 197.8 m2 g-1 and average pore size from 1.55 to 3.4 nm. The drug release

behaviour of HCSNs with different shell thicknesses is investigated using doxorubicin as the model drug. A delay in

the drug release for *300 min is successfully achieved by employing HCSNs with enhanced thickness of 60 nm.

Application of HCSNs in targeted drug delivery was further supported by the in-vitro cytotoxicity studies carried out

on lung adenocarcinoma cells.

Keywords. Hollow core-shell silica nanoparticles; polystyrene; drug delivery.

1. Introduction

Hollow core-shell silica nanoparticles (HCSNs) have made

notable impact in the field of targeted drug-delivery systems

due to the following properties; tunable morphology, large

specific surface area and biocompatibility [1–3]. Promising

applications of HCSNs also include catalysis, chromato-

graphic separations, membranes, electronic devices and

sensors [2–6]. Various synthesis techniques have been dis-

cussed in the literature to synthesize HCSNs, like sacrificial

template, hydrothermal, sol/gel and sonochemical tech-

niques [7–9].

Sacrificial template method is one of the efficient

methods for the synthesis of nanosized hollow core-shell

silica-structured particles. In this template method, mor-

phology of silica nanoparticles could be tuned by varying

parameters such as size, shape and surface properties of

the template. Thus, sacrificial template methods provide a

flexible route to synthesize HCSNs with tunable mor-

phology. Various templates used during the synthesis of

HCSNs include polystyrene (PS), PS-b-poly(acrylic acid),

cetyltrimethylammonium bromide (CTAB), PS-methyl

acrylic acid, liposomes and ZnSe [7–11]. Among them PS

is a versatile template using which morphology of silica

shell could be improved by varying the surface charge of

the sacrificial template. Surface properties of PS tem-

plates were modified by functionalization of PS such as

carboxyl, amino, sulphonate and nitro functional groups

[7–9]. Yang et al [8] used microspheres of sulphonated

PS to synthesize hollow polyaniline and hollow poly-

pyrrole particles. Sulphonated PS silica composites were

employed as ion-exchange materials, adsorption of metal

ions and solid acid catalysts [10–12]. Liu et al [13]

reported synthesis of hollow porous silica particles using

sulphonated PS-methyl acrylic acid template. Utilization

of sulphonate functionalized PS nanoparticles during the

fabrication of HCSNs for use in the field of drug delivery

requires a detailed study.

The synthesis of monodispersed silica spheres by Stober

method involved hydrolysis-condensation of tetraethyl

orthosilicate (TEOS) in water–ethanol mixture using

ammonia as a catalyst [14]. Stober method was altered by

the use of polymeric template and cationic surfactant to

synthesize nanometer or sub-micrometre-sized hollow silica

spheres [15,16]. Various precursors of silica used during the

synthesis were tetramethoxysilane, tetraethoxysilane and

colloidal silica particles [17–19]. Facile synthesis of HCSNs

by merging the template and Stober methods resulted in the

formation of HCSNs with tunable thickness and narrow

pore size. These features were explored while HCSNs were

Bull Mater Sci (2020) 43:213 � Indian Academy of Scienceshttps://doi.org/10.1007/s12034-020-02209-0Sadhana(0123456789().,-volV)FT3](0123456789().,-volV)

used as drug-delivery vectors [20,21]. HCSNs received

much attention among various drug-delivery systems such

as liposomes, dendrimers, nanotubes and hydrogels

[22–25]. HCSNs were found to have good biocompatibility

and chemical stability which fit the basic requirements of

the targeted drug-delivery systems. The tunable morphol-

ogy of mesoporous silica nanoparticles permitted incorpo-

ration of drug particles and targeted drug delivery to the

sites of disease [26,27].

The synthesis of HCSNs with enhanced shell thickness

using sulphonated PS template, lead to the delayed release

of drug is comparatively less studied in the field of targeted

drug delivery. In this research study, sulphonation of PS is

carried out to improve the surface charge properties of PS.

Sulphonated PS is used as a template to synthesize the

HCSNs with enhanced thickness by modified Stober

method. A detailed investigation is conducted to analyse the

influence of sulphonated PS on variation of surface area and

shell thickness of mesoporous HCSNs. A systematic study

is conducted to determine the drug loading and release

behaviour in controlled form for HCSNs at different shell

thicknesses. Cytotoxicity studies were conducted for

HCSNs, doxorubicin (DOX) loaded HCSNs and DOX on

lung adenocarcinoma (A549) cells.

2. Materials and methodology

2.1 Materials

Styrene (99%), polyvinyl pyrrolidone (PVP, 40,000-

molecular weight), TEOS (98%) and DOX were obtained

from Sigma Aldrich. Sulphuric acid (98%), potassium per

sulphate (KPS, 98%) and CTAB (98%) were purchased

from Loba Chemie. Aqueous ammonia solution (25%) and

ethanol (99%) were procured from Spectrum Chemicals and

Changshu Hongsheng Fine Chemical Co. respectively. All

the chemicals were used as collected without any treatment.

Purified water was obtained from a Millipore purification

apparatus.

2.2 Methods

2.2a Synthesis of sulphonated PS template: PS nanopar-

ticles were synthesized by using emulsion polymerization

method as previously described in the literature [28] by

employing styrene as a monomer, KPS as the initiator

and PVP as a stabilizer. One gram of synthesized PS was

dispersed in a mixture of 0.15 moles of concentrated

sulphuric acid and 27.7 mmoles of water. The reaction

was performed at room temperature for 2 h. Ethanol was

added in excess to resume the sulphonation and stirred

for 30 min. Sulphonated PS nanoparticles were washed 6

times in water by centrifugation at 12,000 rpm for

10 min. The samples were dried in a vacuum drier to

remove the moisture adsorbed.

2.2b Synthesis of HCSNs: HCSNs were synthesized using

sulphonated PS as a template. One gram of synthesized

sulphonated PS template was allowed to disperse in water—

ethanol mixture maintained at a ratio of 4:1 by using

ultrasonication for 30 min. A volume of 10 ml of 5%

solution of CTAB was added to the mixture followed by

magnetic stirring for 3 h. A measure of 0.5 ml of ammonia

was added to the solution preceded by the addition of

TEOS–ethanol mixture in the ratio of 1:1. Magnetic stirring

was continued for 4 h at room temperature. Thus obtained

suspension was aged at room temperature for 24 h. The

precipitate containing silica-coated sulphonated PS was

separated by centrifugation (10,000 rpm for 15 min) and

washed five times with water. Resulting samples were dried

in a vacuum drier to remove moisture content and calcined

at 550�C for 4 h at a heating rate of 1�C min-1 to remove

sulphonated PS core.

2.2c Loading and release studies: A 50 mg of HCSNs

were dispersed in 100 ppm solution of DOX by ultrasoni-

cation for 3 min and stored at 4�C for 48 h. DOX-loaded

HCSNs were separated by centrifugation (at 12,000 rpm for

15 min) and dispersed in phosphate-buffered saline (PBS)

of pH-7.4. The samples were stirred mildly at 37�C. A

volume of 5 ml of solution was withdrawn at known time

intervals and centrifuged at 12,000 rpm for 5 min to study

the release behaviour of DOX. The supernatant was anal-

ysed in a UV–visible spectrophotometer at a wavelength of

480 nm and it was poured back to maintain the constant

volume. The release studies were carried out at pH 6 using

phosphate buffer (PB), to study the effect of pH on the

release.

A volume of 1 litre PBS buffer at pH 7.4 is prepared by

dissolving 0.137 mol of NaCl, 2.7 mmol of KCl, 0.01 mol

of Na2HPO4 and 1.8 mmol of KH2PO4 in water using a 1 L

volumetric flask. PB buffer at pH 6 is prepared by mixing

95 ml of 0.1 M KH2PO4 and 5 ml of 0.1 M Na2HPO4 in

water using 1 L volumetric flask.

2.2d In-vitro cytotoxicity assays: The in-vitro cytotoxicity

studies were carried out by 3-(4,5-dimethylthiazol-2-yl)-

2,5-diphenyltetrazolium bromide (MTT) assay method for

HCSNs and SPION embedded HCSNs. Lung adenocarci-

noma (A549) cells were procured from National Center for

Cell Sciences (NCCS), Pune. They were cultured in Dul-

becco’s modified Eagle’s medium (DMEM) supplemented

with 10% fetal bovine serum (FBS) and 1% antibiotic

antimycotic solution. The cells were maintained at 37�Cwith 5% CO2 in a humidified atmosphere. The cells were

seeded onto 96-well microtitre plates at a seeding density of

5000 cells per well. After adherence, they were treated with

different concentrations of the HCSNs samples such as 25,

50, 100 and 200 lg ml-1. MTT assay reagent was added

and incubated at 37�C for 4 h, after 48 h of post incubation

213 Page 2 of 9 Bull Mater Sci (2020) 43:213

period. Formazan crystals formed were solubilized using

dimethyl sulphoxide and absorbance was recorded at

570 nm using a multimode microplate reader (FluoSTAR

Omega, BMG labtech). Percentage viability of the test

compounds was calculated with respect to the cell control.

2.3 Characterization techniques

HCSNs were gold sputtered and observed under a scanning

electron microscope (SEM, JEOL-JSM 6380 LA) to study

the surface morphology. HCSNs were dispersed in ethanol

by ultrasonication and a few drops were added to the carbon

coated copper grid prior to visualization in transmission

electron microscopy (TEM, JEOL JEM-2100). Horiba (SZ-

100) instrument was used to find out the zeta potential and

particle size, by dispersing the samples in distilled water by

ultrasonication. Zeta potential was determined at pH 7 and

average of three measurements were calculated with the

standard deviation and conductivity. The autocorrelation

function for dynamic light scattering is given as

G2 sð Þ ¼ I tð Þ � I t þ sð Þ; ð1Þ

where G2(s) is the autocorrelation function, I(t) representsscattering light intensity and s is the short time difference.

Surface area analyser (Quantachrome Corporation,

NOVA1000) was used to study N2 adsorption/desorption

isotherms of the HCSNs. The specific surface area and

the pore-size distribution were calculated by Brunauer–

Emmett–Teller (BET) and Barrett–Joyner–Halenda (BJH)

methods, respectively. The sample preparation involved

degassing of samples under a vacuum at 450�C for 8 h and

the analysis was performed at 77 K. Fourier transform

infrared spectra of PS, sulphonated PS, silica-coated sul-

phonated PS and HCSNs were recorded in Thermonicolet

Avatar-370 using KBr pellet method. Thermogravimetric

analysis (TGA) was carried out using TG/TGA instrument

(Hitachi-6300) with a heating rate of 10 K min-1 from

room temperature to 750�C in the presence of air. The DOX

release behaviour of HCSNs was observed in UV–visible

spectrophotometer (Hitachi, U-2900).

3. Results and discussion

3.1 Synthesis and characterization of HCSNs

FTIR absorption spectra of PS, sulphonated PS, silica-

coated sulphonated PS and HCSNS are presented in

figure 1. The peaks at 696 and 754 cm-1 correspond to

vibrations (C–H) of the benzene ring and those at 1493,

1451 and 1600 cm-1 are attributed to vibrations of benzene

ring (C–C) of PS [29,30] (figure 1a). Sulphonation of PS is

confirmed by the occurrence of a strong absorption band at

1170 cm-1 (figure 1b) corresponding to the sulphonic

group (–SO3H) [13,31]. Figure 1f shows the chemical

structure of sulphonated PS according to Mulijani et al [32].In figure 1c, a peak at 951 cm-1 reveals that CTAB is

coated on sulphonated PS nanoparticles [33]. Figure 1d

shows FTIR spectrum of HCSNs. The sequence of

absorption bands comprising of 1093 and 804 cm-1 belong

to Si–O–Si asymmetric and symmetric stretching vibration.

The presence of silanol groups (Si–OH) on the surface is

confirmed by the peak at 962 cm-1. The oxygen deforma-

tion vibration (Si–O–Si) is observed from the band at

460 cm-1 [34]. The bands due to alkyl groups vanish in

HCSNs in the range of 2800–2900 and 2900–3000 cm-1

which is in agreement with the complete removal of sul-

phonated PS core during the process of calcination [35].

HCSNs have chemical structure with Si–O–Si and Si–OH

bonds as given in figure 1e [36]. Si–OH bonds face towards

the surface of the shell.

SEM images of PS and sulphonated PS are displayed in

the figure 2a and b which show monodispersity and spher-

ical morphology of the samples are retained even after

sulphonation. PS and sulphonated PS showed an average

particle size of 140 and 160 nm, respectively (figure 2c and

d). An increase in the size of sulphonated PS could be due

to the increase in hydrodynamic diameter of the particles, as

there was an increase in the number of functional groups on

the surface of the sulphonated PS. The zeta potential anal-

ysis results of PS and sulphonated PS are shown in table 1.

There was *11.7 mV decrease in the zeta potential after

the sulphonation of PS, attributed to the presence of sul-

phonic groups.

The TEM images of HCSNs synthesized by altering the

ratio of PS/TEOS (1:1, 2:3, 4:7 and 1:2) are shown in fig-

ure 3. Silica nanoparticles exhibited a structure containing

hollow internal core and thick shell confirmed by the TEM

images (figure 3a–d). High-resolution TEM images were

also captured for the HCSNs samples to observe the pres-

ence of the silica shell (figure 3a–c).

The concentration of silica precursor and surface charge

of template play a key role in tuning the shell thickness

of HCSNs. During the synthesis step of HCSNs, TEOS

hydrolysed and deposited on the surface of CTAB-coated

sulphonated PS. The thickness of silica shell was found to

increase from 20 to 60 nm with increase in quantity of

TEOS from 1 to 1.75 g while PS/TEOS ratio varied from

1:1 to 4:7 (figure 3a–c). When the concentration of TEOS

was enhanced, additional amount of silica was deposited on

the surface of sulphonated PS which enhanced the thickness

of the shell [28,37]. Shell thickness of HCSNs synthesized

using the sulphonated PS template was found to be higher,

compared to that of HCSNs synthesized using PS template

[28]. This phenomenon accounted to the increase in

attractive force between the positively charged CTAB and

negatively charged silica ions.

The presence of sulphonic acid group enhanced the

negative charge on the surface of PS as confirmed by zeta

potential analysis. This phenomenon might have enhanced

the deposition of CTAB on the surface of sulphonated PS.

Bull Mater Sci (2020) 43:213 Page 3 of 9 213

Thus, a higher thickness of silica shell was attained using

sulphonated PS as the template. The silica shell thickness

was found to increase with increase in the acidity of acidic

functional group [13]. Further increase in the concentration

of TEOS (PS/TEOS ratio of 1:2) lead to the evolution of

undesirable free solid silica particles due to the fast rate of

formation of silica as observed in figure 3d. PS templates

were found to be inadequate to capture all silica nanopar-

ticles on the surface [38].

TGA analysis was conducted to determine the thermal

stability of the synthesized nanoparticles. TGA curves for PS,

sulphonated PS, silica-coated sulphonated PS andHCSNs are

represented in figure 4. Weight loss observed at lower tem-

peratures below 100�C was due to the evaporation of water

absorbed on the surface of the particle [39]. Initial loss in

weightwas found to bemaximum in case ofHCSNs due to the

entrapment ofmoisture on surface pores. Sulphonated PS and

silica-coated sulphonated PS displayed a quick drop at

*340�C and no weight loss was observed above 550�C.Thus, the absence of any remnant sulphonated PS particles

along with silica was confirmed at higher temperatures

([550�C). Twenty-eight per cent of silica residuewas presentafter 550�C, when silica-coated sulphonated PS was ther-

mally decomposed. TGA graph of silica did not show any

decomposition after 100�C which confirmed the absence of

any polymer residue in the HCSNs (figure 4).

N2 adsorption/desorption studies were performed on

HCSNs with varied shell thicknesses (Ts1, Ts2 and Ts3) as

shown in figure 5. The N2 adsorption/desorption isotherms

displayed a type-IV curve, indicating mesoporous nature of

the shell with hollow internal cavity [40–42]. Hysteresis was

noticed at the relative pressure range from0.4 to 1 for samples

Ts1, Ts2 and Ts3 (figure 5). The nature of hysteresis loop

agreed with H4 type as observed from the BET isotherms

[43]. The average pore sizes were determined by using BJH

method and found to vary from 1.55 to 3.4 nm with increase

in shell thickness from 20 to 60 nm (table 2). Specific surface

area of HCSNs was found to reduce from 644.1 to 197.8 m2

g-1 with increase in shell thickness of silica from 20 to 60 nm

(table 2). When the concentration of TEOS was increased,

the thickness of the silica shell increased while more number

of silica particles deposited on the shell quickly. Thus, a rapid

condensation of silica particles on the surface occurred. It

may lead to the formation of a less rigid network of silica

particles with a higher pore size of 3.4 nm.

The surface of the sulphonated PS template was nega-

tively charged due to the presence of sulphonic acid group

confirmed by FTIR analysis (figure 1). The surface of sul-

phonated PS was coated employing cationic surfactant

CTAB by electrostatic force. CTAB acted as a major

ingredient in the formation of HCSNs. Self-assembly of

TEOS on sulphonated PS was guided by CTAB. Further,

Figure 1. FTIR spectra of (a) PS, (b) sulphonated PS, (c) silica-coated sulphonated PS, (d) HCSNs, (e) HCSNs chemical structure [36],

(f) sulphonated PS chemical structure [32].

213 Page 4 of 9 Bull Mater Sci (2020) 43:213

TEOS was allowed to hydrolyse in the basic condition

forming siliceous micelles with negative charge. Calcina-

tion of silica-coated sulphonated PS leads to the formation

of HCSNs and complete removal of polymer template. A

higher thickness of silica shell was achieved while sul-

phonated PS template was used [13,16].

3.2 DOX loading and release studies

The promising application of HCSNs as a drug-delivery

vector was investigated by using DOX as the drug. Loading

capacity and encapsulation efficiency were determined

according to the literature [44]. The loading capacities of

samples Ts1, Ts2 and Ts3 were calculated according to the

equation (1) and were found to be 1.31, 1.11 and 1.06 wt%,

respectively. Since the core sizes of HCSNs were nearly the

same, similar values of loading capacities were observed.

Encapsulation efficiencies were determined for samples

Ts1, Ts2 and Ts3 and were found to be 45.4, 39.1 and 37.4

wt%, respectively. The decrease in encapsulation efficien-

cies of samples Ts1, Ts2 and Ts3 could be due to the

reduction in specific surface area of samples from 644.1 to

197.8 m2 g-1 [45]. The reduction in specific surface area

could be due to the reduction in number of surface pores

with increase in the thickness of the silica shell when sul-

phonated PS was used as the template.

The cumulative DOX release plot of HCSNs samples

synthesized using PS template and sulphonated PS template

are shown in figure 6 at pH 6 and 7.4. The drug release

mechanism for rigid mesoporous systems were noted to be

diffusion controlled [46]. Initial burst release of drug upon

contact with PBS was highest for sample with lowest

Figure 2. SEM images of (a) PS, (b) sulphonated PS. Particle-size distribution from dynamic light scattering (c) PS, (d) sulphonatedPS.

Table 1. Zeta potential analysis of PS and sulphonated PS.

Sample

Zeta potential

(mV)

Standard

deviation

Conductivity

(mS cm-1)

PS -53.3 0.43 0.064

Sulphonated

PS

-65.0 1.6 0.088

Bull Mater Sci (2020) 43:213 Page 5 of 9 213

thickness 20 nm (Ts1). However sample Ts3 with the highest

thickness showed minimum burst release than the other

samples [47]. The rapid release of drug occurred during initial

15 min and accounted for discharge of drug particles present

on the surface and pore entrance ofHCSNs [48].After 1 h, the

percentage cumulative release ofDOX reached 49.1, 43.8 and

35.2% for samples Ts1, Ts2 and Ts3 (at pH 7.4), respectively.

It was observed that the sample Ts1 exhibited the highest

rate of DOX release and sample Ts3 exhibited the lowest

rate of DOX release. Influence of average specific surface

area of HCSNs and thickness of the silica shell on the drug

release rate was the prominent reason behind variation in

the DOX release for HCSNs samples (Ts1, Ts2 and Ts3).

Sample Ts1 with the highest specific surface area and lowest

thickness (table 2) had shorter mesopore length which

caused the rapid release of DOX. However, reduced release

rate was observed for sample Ts3, which had the lowest

Figure 3. TEM images of HCSNs synthesized by varying PS/TEOS ratio (a) 1:1, (b) 2:3, (c) 4:7, (d) 1:2.

Figure 4. TGA plot of PS, sulphonated PS, silica-coated

sulphonated PS, HCSNs.

213 Page 6 of 9 Bull Mater Sci (2020) 43:213

specific surface area and highest thickness (table 2)

[28,42,45]. Further, steady release of drug was observed for

300 min as shown in figure 6. HCSNs samples synthesized

using PS template were found to have lower thickness

(15–30 nm), hence showed steady release up to 200 min as

reported earlier [28]. However, sample Ts3 achieved greater

delay in the initial burst and steady release of DOX for

prolonged time compared to other samples.

pH of the drug release medium is considered to be one of

the important parameters which affect the release of drug

from the carrier [49]. pH of the cancer cell was found to be

lower than the normal cells. Effect of pH of phosphate

buffer was studied for HCSNs samples (Ts1, Ts2 and Ts3)

by maintaining pH of the release medium at 6 and 7.4. It

was found that at higher pH of 7.4, drug release was

comparatively less (30.5% in 15 min for Ts3). It could be

the result of poor solubility of drug at higher pH. At lower

pH of 6, higher release of drug (40.7% in 15 min for Ts3)

was observed. The drug release followed similar trend at pH

6 and 7.4 as shown in the plot (figure 6). The quantity of

drug released at pH 6 was found to be higher than that at pH

7.4 [42].

Figure 5. N2 adsorption/desorption isotherm and pore-size distribution for sample (a) Ts1, (b) Ts2, (c) Ts3.

Table 2. Variation of specific surface area and pore size with thickness of silica shell.

Sample PS/TEOS ratio Shell thickness (nm) Average pore diameter (nm) Specific surface area (m2 g-1)

Ts1 1:1 20 1.55 644.1

Ts2 2:3 40 2.78 391.5

Ts3 4:7 60 3.40 197.8

Bull Mater Sci (2020) 43:213 Page 7 of 9 213

3.3 In-vitro cytotoxicity studies

The in-vitro cytotoxicity studies were conducted on lung

adenocarcinoma (A549) cells by MTT assay method to

assess the possible usage of HCSNs in targeted drug-

delivery systems. Figure 7 shows the results of cytotoxicity

studies for HCSNs, DOX-loaded HCSNs and DOX. Death

of cancer cells was found to increase with the increase in

concentration of DOX-loaded HCSNs. Thus, the percentage

of viable cells reduced from 60.2 to 13.9% as the concen-

tration of HCSNs was increased from 25 to 200 lg ml-1 as

shown in figure 7. The cytotoxicity assays of blank HCSNs

were conducted as control in the concentration range from

25 to 200 lg ml-1. The results confirmed that HCSNs did

not cause any toxicity at lower concentrations [42,50]. The

viability of A549 cells at higher concentration of HCSNs

(200 lg ml-1) was found to be high at about 96% and

demonstrated the good biocompatibility.

4. Conclusions

HCSNs of particle size *160 nm were successfully

synthesized using sulphonated PS as the sacrificial tem-

plate. Sulphonation of PS has modified the negative

charge on the surface of PS from -53.3 to -65 mV.

Thickness of silica shell was tailored from 20 to 60 nm

by altering the concentration of TEOS in PS/TEOS ratio

from 1:1 to 4:7. HCSNs synthesized with sulphonated PS

template showed higher thickness than those synthesized

using PS template due to the enhanced surface charge of

the sulphonated PS template. Hence, dependence of

thickness of silica shell on the surface charge of the

template was obvious. Specific surface area of HCSNs

decreased remarkably from 644.1 to 197.8 m2 g-1, with

increase in the thickness of the silica shell. The delay in

DOX release about 300 min was achieved by monitoring

key properties of HCSNs like shell thickness, specific

surface area and average pore size (1.55–3.4 nm). In-vitrocytotoxicity studies also mark them suitable for the

application in targeted drug delivery.

Acknowledgements

We acknowledge the funding was provided by Council of

Scientific and Industrial Research, India (Grant No. 22/646/

13/EMR-II).

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