Biocompatibility of a sonochemically synthesized poly(N-isopropyl acrylamide)/silica nanocomposite

Abstract

A novel inorganic–organic core–shell nanocomposite, poly(N-isopropyl acrylamide) coated silica nanoparticles (PNIPA-g-FSNP), has been synthesized sonochemically for the first time following the principles of green methods. The sonochemical method eliminates the requirement for organic solvents, cross-linkers and hydrophobic agents used in the conventional miniemulsion technique. The method not only increases the reaction rate, but also leads to regular size distribution of the products. The composites are characterized by TEM, SAED, EDX, DLS, Z potential, FTIR and TGA. The biocompatibility of the synthesized materials has been studied in the light of protein adsorption, histopathology, haematological and non-thrombogenic property for probable use in the field of safe drug delivery. Bare silica nanoparticle has drug loading capability, but it lacks haemo-compatibility, tissue specificity and site directing ability.

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1. Introduction

Polymer silica nanocomposite gained attention in recent years for the development of specific material for biomolecular adsorption, enzyme immobilization, temperature responsive ibuprofen release, bone tissue engineering, stimuli-responsive nano-valves, thermo-sensitive vehicles for cellular imaging, bio-catalysis, optoelectronic devices^1–4 and synthesis of mesoporous carbon.

Silica nanoparticles possess high thermal stability, chemical versatility, drug loading capacity and non-biodegradability that are crucial for many biomedical applications such as in the formulation of artificial implant, and storage and release of drugs.⁵ However, they have the tendency to absorb serum albumin (blood protein), which is responsible for their poor haemo-compatibility.⁶ Due to inertness they lack tissue specificity and site directing ability in the case of drug delivery. But this bottleneck may be overcome by encapsulating them with a suitable bio friendly polymer.⁶

Poly(N-isopropyl acrylamide) (PNIPA) is a popular thermo-sensitive polymer and it has lower critical solution temperature (LCST) at about 32 °C, which is close to physiological temperature. PNIPA undergoes a thermo-induced conformational change from the swelled (hydrophilic) state to the shrunken (hydrophobic) state above LCST in water.⁷ So PNIPA may be used as a site specific drug delivery material, cell attachment/detachment matrix and haemostatic agent. Hence, PNIPA along with silica nano particles may attribute many desireable properties for bio-medical applications of the composites. The presence of both hydrophilic (–CONH) and hydrophobic (–CH–(CH₃)₂) groups, makes the PNIPA a beautiful polymeric surfactant.⁸

Recently a considerable amount of works have been reported^7,9 on the synthesis of PNIPA coated silica nanoparticles. Most researches have been focused on PNIPA due to its LCST, however, as far as we are aware, few people pay attention to its biocompatibility, which is an essential criterion for biomedical application. Yang et al.⁷ prepared PNIPA coated mesoporous silica nanoparticles (MSN@PNIPAM) by a ‘grafting form' method for cellular imaging. Liu et al. prepared PNIPA grafted colloidal silica through conventional ‘chemical technique’.⁹ Literature survey reveals that nobody has attempted to prepare poly(N-isopropyl acrylamide)/silica (PNIPA-g-FSNP) nanocomposite by ‘sono-chemical technique’. The detail bio-compatibility studies (histopathology and hematology) of the nanocomposite have not been done yet.

Ultrasonic irradiation provides unusual reaction conditions (extremely high temperatures and pressures within micro-droplet reactors), which generates free radicals (H˙ and OH˙) in green aqueous medium.¹⁰ The micro-droplet reactors created by ultra-sound facilitate the formation of nanocomposites. Compared to traditional methods, the main advantages of the sonochemical process are milder reaction conditions, shorter reaction times and higher yields of non-agglomerated nanocomposites.¹¹

Here we report the synthesis of PNIPA coated silica nanocomposite in aqueous medium following the principle of green synthesis. The polymerization occurs by the ultrasound irradiation on the surface of silica nanoparticles. No cross-linker is used for the polymerization. The silica surface has the crucial role on the polymerization. The biocompatibility of the nanocomposite has been evaluated by means of several techniques.

2. Experimental section

2.1. Materials

2.1.1. Chemicals and reagents. Silicon dioxide nanopowder, 10–20 nm (TEM) (SNP, Sigma-Aldrich, Steinheim, Germany), ethanol (EtOH, Merck, Mumbai, India), N-isopropyl acrylamide (NIPA, Sigma-Aldrich, St. Louis, USA), potassium peroxodisulphate (KPS, Merck, Mumbai, India), bovine serum albumin (pH 7, BSA, Himedia, Mumbai, India), Bradford reagent (Sigma-Aldrich, Steinheim, Germany), sodium dodecyl sulphate (SDS, Merck, Mumbai, India), 3-(trimethoxysilyl) propyl methacrylate (TPM, Sigma-Aldrich, St. Louis, USA) were used as received.

2.1.2. Biological materials.
Experimental animals and their maintenance. Randomly bred male Swiss albino mice weighing 25 ± 5 g (8 weeks of age) were procured from the animal facilities of Dey's Medical (Kolkata, India). The experimentations on the animals for this study received prior approval of the Institutional Animal Ethics Committee and met the standard laid down by the Government of India.

2.2. Instruments

The FTIR spectrum was recorded using KBr pellets by Shimadzu-8400S spectrometer. TGA and DTA traces were obtained using a PerkinElmer thermal analyzer (STA-600) in nitrogen at a heating rate of 10 °C min⁻¹. A sonicator (Branson-1510) was used to obtain ultrasound of 40 kHz. Centrifuge machine (Sigma 30 KS) was used for centrifugation. Particle size and its distribution were recorded using Transmission Electron Microscope (TEM) (JEM-2100, 200 kV, Jeol) and dynamic light scattering (DLS) measurements and zeta potential determination were carried out in Malvern instrument. UV-Vis-NIR spectrophotometer (Shimadzu Model UV-PC) is used for determination the concentration of BSA protein in BSA protein adsorption test. A vacuum oven of Eastern Instrument (International, India) was used for drying the samples. The counting was done on a hemocytometer (Feinoptik, Blakenburg, Germany). The stained slides were examined manually under an optical microscope (Prime, Dewinter Optical Inc., Italy).

2.3. Synthesis of poly(N-isopropyl acrylamide) coated silica nanoparticles

Synthesis of poly(N-isopropyl acrylamide) coated SNP was done by ‘grafting from technique’⁸ through functionalization of the later. SNP (5 g) was mixed well with the solution of coupling agent (5 ml TPM in 50 ml ethanol) by a magnetic stirrer and then reflux the solution in an oil bath for 4 h. The functionalized product was separated by centrifugation and dried to a constant weight using a vacuum oven. The TPM : SNP (weight ratio) was kept at 1 : 1.

In the second step, the functionalized product (FSNP) was subjected to graft co-polymerization with NIPA using the ultrasound (42 kHz) induced mini emulsion technique⁶ (Scheme 1) at 60 °C. The emulsion contains FSNP (5 g), NIPA (8 g), double distilled H₂O (500 ml), SDS (8 g) and catalytic amount of KPS. The grafted product (PNIPA-g-FSNP) was obtained as a stable colloidal solution.

Scheme 1 Synthetic route for the preparation of PNIPA-g-FSNP.

2.4. Determination of percentage of grafting

The percentage of grafting was calculated from the TGA traces of the product and comparing it with that of FSNP.⁶ The working formula used in computing percentage of grafting is: percentage of grafting = (weight of the polymer grafted/weight of the FSNP) × 100.

2.5. BSA protein adsorption test

The synthesized material (PNIPA-g-FSNP) (1 mg ml⁻¹) was suspended in aqueous solution of Bovine Serum Albumin (BSA) (1.4 mg ml⁻¹) at pH = 7.0. The mixtures were then shaken at room temperature for 2 h to reach adsorption equilibrium. The nanocomposite was removed by centrifugation and the supernatants were analyzed for BSA using Bradford's method^12,13 with UV-Vis spectrophotometer at wavelength 595 nm. The similar experiment was done with bare SNP. All the experiments were performed three times and the average value of BSA protein adsorption is taken.

2.6. Determination of LD₅₀

For determination of the median tolerance limits for SNP the laboratory-acclimatized healthy mice were divided into two groups each containing ten individuals. One group of mice was injected different dosages of SNP at their peritoneal cavity. The control group of mice was injected sterile physiological saline and maintained throughout the experimental period under identical experimental conditions. Behaviour and other responses of the animals to treatment were carefully noted. Observations were made 24 h post treatment and the number of dead mice was recorded. Whether any bioassay animals died due to factors other than exposure to the test chemicals, corrected mortality was calculated using the Abbott's formula as follows:

Corrected mortality (%) = {(M_obs − M_control)/(100 − M_control)} × 100

The percentage of animal mortality at each dose level was transformed to probits and then LD₅₀ was determined following Miller and Tainter.¹⁴ LD₁ termed as toxicant threshold dose¹⁵ is derived from the linear regression curve of log concentration and probit value of mortality percentage. The sub-lethal dosages were considered which were below the LD₁.

The log concentration of the SNP corresponding to empirical probit value 5 was determined as log LC₅₀ value which was converted to LC₅₀. Similarly LC₁ value is determined from the linear regression equation using the probit value 0.1. Numbers of mice used for each control and treated concentrations for SNP are 10.

2.7. Experimentation for biological studies

Three sub-lethal doses (2.5, 5.0 and 10.0 mg kg⁻¹ body weight) of PNIPA-g-FSNP and SNP were selected. The selected doses of each of the nano composite and the nanomaterials were administrated to ten numbers of mice through intraperitoneal route once a day for an experimental period of 7 days. The control group was maintained by injecting same volume of 0.7% sterile physiological saline following the same schedule.

2.8. Haematological study

To assess the biocompatibility of the synthesized materials, hematological variables were studied in terms of clotting time (CT), total erythrocyte count (TEC), total leucocytes count (TLC), haemoglobin content (Hb), haematocrit (Hct) and differential leucocytes count. Some derived parameters were also worked out such as mean corpuscular volume (MCV), mean corpuscular haemoglobin concentration (MCHC), mean cell haemoglobin (MCH).

2.9. Determination of clotting time (CT)

Clotting time of blood was estimated by capillary glass tube method.⁶

2.10. Total erythrocyte count (TEC) and total leucocytes count (TLC)

Blood of the anaesthetized mice was drawn with a sterile 2 ml heparinised syringe through a cardiac puncture made by direct insertion of the needle. Blood was then collected in small glass vials and mixed with 20 μl of 2.7% EDTA solution. Total erythrocyte count (TEC) and total leukocyte count (TLC) were done following the method of Estridge et al.¹⁶ The counting was done on a hemocytometer (Feinoptik, Blakenburg, Germany) and expressed as:

Number of RBC/mm³ = N_r × 10 000

Number of WBC/mm³ = N_w × 500

where, N_r is the total number of red blood cells counted in five squares of the hemocytometer and 10 000 is the factor obtained after taking into consideration the initial dilution factor and N_w denotes the total number of white blood cells counted in four squares of the hemocytometer and the factor obtained after taking into consideration the initial dilution factors was 500.

2.11. Estimation of hemoglobin by cyanmethemoglobin method

The blood haemoglobin content (Hb) was analyzed following the cyanmethemoglobin method¹⁷ using Darbkin's fluid (Qualigens diagnostics kit, Mumbai, India) in a UV-1601 Shimadzu spectrophotometer at 540 nm wavelength.

2.12. Measurement of haematocrit

Haematocrit of blood was estimated by micro capillary method. Heparinised blood was drawn into capillary tubes and centrifuged at 1000 rpm at 40 °C for 10 minutes. Haematocrit or packed erythrocytes percent was estimated following G. Nardini et al.¹⁸

2.13. Measurement of absolute corpuscular values

The mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH) and mean corpuscular hemoglobin concentration (MCHC) were calculated from TEC, Hb and Hct values according to the method of Gipp et al.¹⁹ using the following formulae:

MCV (cubic micra/femtoliter) = (Hct (%) × 10)/(RBC (million per cubic mm))

MCH (pictograms/pg) = (Hb (g per 100 ml)) × 10/(RBC (million per cubic mm))

MCHC (%) = (Hb (g per 100 ml) × 100)/(Hct (%))

2.14. Differential WBC count

A small drop of blood of the anaesthetized mice was taken directly on the centre of a cleaned grease free glass slide. A blood film was drawn immediately with a clean smooth edged slide placed at an angle of 45 °C to the slide and kept for air dry. The air dried slides were stained with Leishman's stain.²⁰ The stained slides were examined under an optical microscope (Prime, Dewinter Optical Inc., Italy) and relative percentage of leukocyte types was calculated.²¹

2.15. Histopathology

Both the control and treated mice were anesthetized with ether and then autopsied for collection of tissues. The kidney, liver, spleen and lung were dissected out and fixed in Bouin's fluid for 24 h. After a thorough wash in distilled water the fixed tissues were dehydrated through a graded ethyl alcohol series and embedded in paraffin. The tissue sections of 5 μm thickness were prepared with a rotary microtome and stained with haematoxylin and eosin. The slides were examined (40×) under an optical microscope (Prime, Dewinter Optical Inc., Italy) and photomicrographs were taken.

2.16. Statistical analysis

Except the percentage values, all results are expressed as mean ± standard error of mean (S.E.M.). Data was analyzed statistically with one-way ANOVA followed by Tukey HSD Test. The values of P < 0.05 and P < 0.01 were considered significant. All the statistical analyses were performed using SigmaStat for Windows version 2.03. Graphical representations were done with Microsoft Office Excel 2008.

3. Results and discussion

3.1. Synthesis of poly(N-isopropyl acrylamide) grafted silica nanoparticles

PNIPA coated SNP were synthesized through an ultra-sound assisted chemical process in aqueous neutral medium (Scheme 1) minimizing additives like cross-linker, organic solvents, hydrophobic agents, initiator, etc. The grafted product (PNIPA-g-FSNP) was obtained as stable colloidal solution. The stability of the synthesized solution was tested by keeping the solution one month in a sealed conical flask.

Polymerization of NIPA was carried out both in presence and absence of FSNP. It was observed that graft polymer and homopolymer was formed in presence and absence of FSNP respectively. Ultrasound produces hydrogen and hydroxyl radicals (H˙/˙OH), which in turn leads to FSNP radical (eqn (1)). FSNP radical ultimately produces PNIPA-g-FSNP (eqn (2)).

3.2. Particle size and its distribution (TEM & SAED and DLS & zeta potential)

The samples for TEM images were prepared by placing a drop of suspension onto a standard carbon coated copper grid and then dried in air. TEM images along with SAED (selective area electron diffraction) of PNIPA-g-FSNP are presented in Fig. 2. The growth of SNP is due to encapsulation by the polymer. Thus TEM indicates the core–shell structure of the hybrid material with nanosilica core and polymer shell (average width 5–10 nm). The SAED pattern in the Fig. 1b shows several concentric rings, which further confirm the encapsulation of the polymer on to the FSNP.⁶ Two major observations were made from TEM and SAED study, namely (i) formation of nano sized composite having organic and inorganic origin and (ii) core–shell structure of the composite.

Fig. 1 Linear regression curve of log concentration of SNP and probit value of mortality percentage of mice after 24 h exposure to SNP.

Fig. 2 (1) TEM image (a) and SAED (b) of PNIPA-g-FSNP. (2) Particle size distribution of PNIPA-g-FSNP (DLS).

For DLS and zeta potential studies the colloidal solution of PNIPA-g-FSNP was diluted at about 0.5 mM (roughly calculated by SNP concentration taken at the time of preparation). The sizes (diameter) of the grafted FSNP were found to be in the range 40–120 nm, which is larger, compared to SNP (10–20 nm). The maximum populations of the sizes of the nanomaterials were found at the range of 60–80 nm. The zeta potential of SNP and PNIPA-g-FSNP were found −25 mV and −36.1 mV respectively. The increase in the surface charge (negative value of Z-potential increases) indicates the successful grafting of PNIPA on to the surface of SNP.

3.3. FTIR analysis

The FTIR spectra of SNP, FSNP and PNIPA-g-FSNP are shown in Fig. 3. An inspection of the spectra reveals that two new peaks (at 2926 and 1639 cm⁻¹) are appeared in the spectrum of FSNP, which are absent in the spectra of SNP. The new peak may be attributed to the methylene and carbonyl groups of TPM. Thus the FTIR spectrum indicates the functionalization of SNP by TPM. Similarly, two additional bands are observed at 1525 and 1435 cm⁻¹ in the spectrum of PNIPA-g-FSNP, which are absent in the spectrum of both SNP and FSNP. The two additional bands along with the band at 1639 cm⁻¹ may be assigned to amide group of PNIPA.^6,9 The appearance of the characteristics peaks of polyamide confirms the grafting of PNIPA onto FSNP.

Fig. 3 FTIR spectra of SNP, FSNP and PNIPA-g-FSNP.

3.4. Thermo-gravimetric analysis

Fig. 4 compares the TGA (thermo-gravimetric analysis) traces of SNP, FSNP and PNIPA-g-FSNP. SNP undergoes two stage weight losses while its derivatives exhibit three stage weight losses. The first stage weight loss of all the materials (from 40 °C to 100 °C) is due to the loss of water. The product FSNP (weight loss = 27.0784%) shows better thermal stability than PNIPA-g-FSNP (weight loss = 62.33%). The less thermal stability of the PNIPA-g-FSNP compared to FSNP may be due to easy thermal degradation of an organic polymer, PNIPA compared to inorganic polymer, SNP. The TGA traces reveal that FSNP is less stable than SNP. It is due to the presence of organic moiety (TPM). The TGA curves suggest the successful grafting of PNIPA onto SNP. From the percentage of weight loss data we may determine the percentage of grafting of PNIPA onto SNP. The grafting percentage of PNIPA onto SNP is 48.34.

Fig. 4 TGA traces of SNP, FSNP and PNIPA-g-FSNP.

3.5. Energy-dispersive X-ray spectroscopy (EDX) analysis

The energy-dispersive X-ray (EDX) spectra of SNP and polymer coated SNP (PNIPA-g-FSNP) are shown in Fig. 5a and b respectively. The successful grafting of PNIPA onto the FSNP is evident from the presence of the carbon and nitrogen absorption peaks (Fig. 5b), which are absent in the EDX of SNP (Fig. 5a). Here it is noteworthy to mention that the nitrogen peak has been merged with that of oxygen in the present case. Similar type of merging in EDX was also observed by²² Liu et al. The weight percentage and atom percentage justified the proposed structure of the synthesized material.

Fig. 5 EDX spectra of SNP and PNIPA-g-FSNP.

3.6. BSA protein adsorption test

The result of BSA protein adsorption test shows that extent of BSA protein adsorption is lower in the case of PNIPA-g-SNP (236 mg g⁻¹) than bare SNP (609 mg g⁻¹). The decrease in BSA protein adsorption signifies the biocompatibility¹³ of the material. Although the protein adsorption decreased, it restores its ability to bind with the albumin which is one of the major components of vertebrate blood. Weak interaction of PNIPA coated silica nanoparticle with blood protein gives it an amphiphilic property which would be helpful to transport the substance at the tissue level.⁶

3.7. Haematological study

The values of haematological parameters (TEC, Hb and Hct) decreased gradually with the increase of SNP dose and became significant (P < 0.05) beyond 2.5 mg kg⁻¹ (Fig. 6). The differential counts above threshold level (2.5 mg kg⁻¹) of SNP (Fig. 7) showed that the percentage of monocytes, eosinophils and basophils decreased, while neutrophils and lymphocyte counts increased significantly compared to control. MCV, MCH and MCHC values (Fig. 6) increased, but CT decreased meaningfully (P < 0.05) at the SNP doses above 2.5 mg kg⁻¹. Quite interestingly, the haematological variables did not change and remained within the normal ranges in the case of PNIPA-g-FSNP.

Fig. 6 Blood parameters in PNIPA-g-FSNP and SNP treated mice at the doses of 2.5 mg kg⁻¹, 5 mg kg⁻¹ and 10 mg kg⁻¹ body weight: (a) clotting time (CT) of blood, (b) total erythrocytes count (TEC), (c) haematocrit (Hct), (d) haemoglobin concentration (Hb). Data are analyzed statistically by one-way ANOVA followed by Tukey HSD Test. The ‘a’ indicates significant difference (P < 0.05) between treated groups and control group.

Fig. 7 Total leucocytes count (TLC) and differential count of WBCs in mice treated with PNIPA-g-FSNP and SNP. Data are analyzed statistically by one-way ANOVA followed by Tukey HSD Test. The ‘a’ indicates significant difference (P < 0.05) between treated groups and control group.

The hematological study suggests the development of an anemic condition in SNP treated mice. Reduction in TEC concomitant with depletion of haematocrit value in blood profile points towards aplastic nature of the anaemia. Furthermore, the increased MCH value confirms that the anaemia is not hypochromic. The decrease in haematocrit value may be attributed to total cell depletion in peripheral blood. Probably, the presence of SNP disturbs the steady state mechanisms of blood formation (haemopoiesis).²³ Neutrophils and lymphocytes are increased in the circulation as an effort to evoke a primary defense mechanism and secondary immunity against the foreign SNP respectively. PNIPA-g-FSNP is able to overcome both the primary and secondary immune mechanism which entails about it biocompatibility.

3.8. Histopathology of liver, kidney, spleen and lungs

Liver. Although, no marked alteration was observed in the histological architecture of the liver from mice treated with low level of SNP (2.5 mg kg⁻¹), a remarkable disorganization of hepatic tissue architectures were evident (Fig. 8f and g) at higher dosages (above 2.5 mg kg⁻¹). The hepatic trabecules or chords were markedly affected, sinusoids were obscured and endothelial lining of the central vein were disrupted. Nuclear condensation and pyknosis in the hepatic parenchyma were also evident (Fig. 8f). Severity of hepatotoxicity was further aggravated particularly in the mice treated with 10 mg kg⁻¹ SNP. It also exhibited weight diffused hepatic hydropic degeneration (HD) characterized with vacuolated and necrotic hepatic parenchyma (Fig. 8g). Interestingly, histological architecture of the liver was normal in PNIPA-g-FSNP treated mice.

Fig. 8 Histology of liver of mice (H&E): (a) control, (b) 2.5 mg kg⁻¹ SNP treated, (c) 5 mg kg⁻¹ SNP treated, (d) 10 mg kg⁻¹ SNP treated, (e) 2.5 mg kg⁻¹ PNIPA-g-FSNP treated, (f) 5 mg kg⁻¹ PNIPA-g-FSNP treated, (g) 10 mg kg⁻¹ PNIPA-g-FSNP treated. Cv, central vein; Hc, hepatocytes; Ss, sinusoids; Fhc, faded hepatocytes.

Kidney. The tissue organisation of kidney of the PNIPA-g-FSNP treated mice exhibited normal appearance of tubular and corpuscular structure. Well-organized renal tubules were seen in the histological preparations of kidney tissue. Kidney contained well-developed glomeruli in tightly packed alignment. Bowman's corpuscles were surrounded by columnar epithelial cells and were provided with sufficient luminal space. However, notable changes were observed in the histological sections of SNP treated mice in a dose dependent manner (Fig. 9f and g). Tubular lumen was dilated but capsular lumen was reduced in the kidney of the treated mice. Glomerular disorganizations and disruption of capsular epithelial lining were also noticed in the SNP treated mice (Fig. 9f and g).

Fig. 9 Histology of kidney of mice (H&E): (a) control, (b) 2.5 mg kg⁻¹ SNP treated, (c) 5 mg kg⁻¹ SNP treated, (d) 10 mg kg⁻¹ SNP treated, (e) 2.5 mg kg⁻¹ PNIPA-g-FSNP treated, (f) 5 mg kg⁻¹ PNIPA-g-FSNP treated, (g) 10 mg kg⁻¹ PNIPA-g-FSNP treated. Glm (glomerulous); Rnt (renal tubules); Cs (capsular space).

Spleen. Under treatment of 2.5 mg kg⁻¹ SNP the mice showed slight hyperplasia and increase in cell size of their spleen. At higher doses of 5 mg kg⁻¹ and 10 mg kg⁻¹ SNP tissue disorganization and rupture of red pulp of the spleen were observed (Fig. 10). In the mice under 2.5 mg kg⁻¹ and 5 mg kg⁻¹ PNIPA-g-FSNP treatment no notable changes in spleen tissue organization was observed but at higher dose of 10 mg kg⁻¹ PNIPA-g-FSNP rupture of red pulp and trabeculae was observed in the spleen.

Fig. 10 Histology of spleen of mice (H&E): (a) control, (b) 2.5 mg kg⁻¹ SNP treated, (c) 5 mg kg⁻¹ SNP treated, (d) 10 mg kg⁻¹ SNP treated, (e) 2.5 mg kg⁻¹ PNIPA-g-FSNP treated (f) 5 mg kg⁻¹ PNIPA-g-FSNP treated (g) 10 mg kg⁻¹ PNIPA-g-FSNP treated. Rp – red pulp, Tr – trabeculae, Wp – white pulp, Rrp – ruptured red pulp, RTr – ruptured trabeculae.

Lung. Histopathological examination on lungs revealed that SNP treatments at three different doses of 2.5 mg kg⁻¹, 5 mg kg⁻¹ and 10 mg kg⁻¹ resulted severe necrosis of parenchymal cells. Alveolar inflammation and very often rupture of the alveoli are accompanied with the histopathology (Fig. 11b–d). Parenchymal hyperplasia and disruption of cell membrane were evident in lungs of those SNP treated groups of mice. Among PNIPA-g-FSNP treated groups of mice, at 5 mg kg⁻¹ and 10 mg kg⁻¹ doses alveolar little inflammation with increased alveolar space was observed without noted hyperplasia (Fig. 11f and g). However, in 2.5 mg kg⁻¹ PNIPA-g-FSNP treated mice quite normal histological architecture was maintained (Fig. 11e) with regular size and arrangement of alveoli and bronchioles.

Fig. 11 Histology of lung of mice (H&E): (a) control, (b) 2.5 mg kg⁻¹ SNP treated, (c) 5 mg kg⁻¹ SNP treated (d) 10 mg kg⁻¹ SNP treated, (e) 2.5 mg kg⁻¹ PNIPA-g-FSNP treated (f) 5 mg kg⁻¹ PNIPA-g-FSNP treated (g) 10 mg kg⁻¹ PNIPA-g-FSNP treated. Al – alveoli, RAl – ruptured alveoli, Br – bronchiole.

Discussion of histopathology. Hepatotoxicity and renal toxicity were evident from the death of hepatic parenchyma and endothelial cells as well as from the death of capsular epithelial cells and glomerular endothelial cells of kidney from SNP treated mice. Chen et al.²⁴ underscored the mechanism of hepatotoxicity of silica nanoparticle in mammalian system where they mentioned that silica nano particles were taken up by the phagocytic Kupffer cells. Silica nanoparticles in turn activate the Kupffer cells to mediate hepatic injury via production of specific bioactive substances. The Kupffer cells immediately respond to an external stimulus by production of, inflammatory cytokine TNF-α, reactive oxygen species (ROS) and NO.^25–27 These bioactive mediators may have harmful effects on hepatocytes. TNF-α induces different mechanisms to initiate apoptosis in hepatocytes that leads to hepatic injury. SNP also resulted in cell death in spleen but the severity is the maximum in lung tissue which is an indication of silicosis. The ROS are capable of causing oxidative damage to major cellular structures, specifically the mitochondria and the plasma membrane^28,29 whereas, NO is known to down regulate cytochrome P450 and to suppress biosynthesis of protein and DNA in liver, and these activities may lead to hepatotoxicity.³⁰ It is known that upon entry into the mammalian system nanoparticles are rapidly labeled by certain plasma proteins, a process that is usually referred to as opsonization.³¹ Opsonized nanoparticles are easily recognized and scavenged by the macrophages like Kupffer cells.³² In the contrary, present study also revealed that PNIPA-g-FSNP treated mice did not exhibit any significant alteration in hematological parameters such as TEC, Hb, Hct, TLC, and differential leukocyte count. Moreover, the histology of liver, kidney lungs and spleen PNIPA-g-FSNP treated mice preserved normal tissue architectures. Severity of pathology is less in PNIPA-g-FSNP treated mice in comparison to bared SNP treated mice. The result thus suggests that coating of silica nanoparticles with biocompatible polymer like the poly-N-isopropyl acrylamide (PNIPA) makes the silica nanoparticles biocompatible when the bare one exhibits toxicity in the biological system. The opsonization rate might be decreased if the particle surface is covered with a biocompatible material (PNIPA); likewise the polyethylene glycol does.³⁰ If opsonization is diminished, the macrophage mediated cell death could be prevented. Our experimental result on PNIPA-g-FSNP mice is in conformity to this view. PNIPA is a thermo-responsive water soluble polymer having lower critical solution temperature of about 32–35 °C, which is nearer to the normal body temperature of human beings. This physical characteristic confers the substance suitability as a biocompatible agent. The PNIPA masked silica nanoparticle can thus be a promising vehicle for the modern approach of targeted drug delivery.

4. Conclusion

PNIPA grafted silica nanoparticles (PNIPA-g-FSNP) leading to novel inorganic–organic core–shell nanocomposite has been synthesized sonochemically following the principle of green chemistry. The sonochemical method eliminates organic solvents, cross-linker and hydrophobic agents used in the conventional miniemulsion technique. The method increases reaction rate and induces regular size distribution. In presence of ultrasound, FSNP radical is produced as the major product along with minor quantities of sulfate radical. On the other hand the reverse is true in absence of ultra sound. As a result sonochemical technique maximizes graft copolymerization and minimizes homopolymerization.

Both silicon and silica nanoparticles are currently considered to be promising vehicles for targeted drug delivery. However, there is a paucity of data about their toxicity in vivo. Our experimental results will supplement the dearth of such information on toxicity of silica nanoparticles in vivo condition. Bare silica nanoparticles induce cytotoxicity in vivo in the tissues like blood, liver, kidney, spleen and lung of mice. Coating of silica nanoparticles with a polymer polyN-isopropyl acrylamide (PNIPA) prevents the cytotoxic property of silica nanoparticles and makes it biocompatible. PNIPA coated silica nanoparticle could be a suitable vehicle for targeted drug delivery. The synthesized material is appeared to be haemocompatible and histologically safe and biocompatible.

Acknowledgements

The authors gratefully acknowledge the financial supports provided by the CSIR (no. 01(2444)/10/EMR-II) and DST (no. SR/S3/ME/0018/2010). The authors also acknowledge SAIF, NEHU, Shillong, India for carrying out TEM imaging.

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