Genipin crosslinked soy-whey based bioactive material for atorvastatin loaded nanoparticles: preparation, characterization and in vivo antihyperlipidemic study

Abstract

The aim of the present study was to explore the lipid lowering potential of soy protein isolate (SPI) and whey protein concentrate (WPC) for the development of novel bioactive material as a drug delivery system for encapsulation of atorvastatin (ATR) in nanoparticles (NPs) and to enhance the antihyperlipidemic potential. SPI-WPC crosslinks were synthesized through amine–amine crosslinking reaction using genipin and characterized by FTIR, mass analysis and degree of crosslinking. ATR loaded NPs were prepared by desolvation process and the optimized formulation (ATR/SPI-WPC NPs-10) was selected on the basis of least particle size (158.1 ± 3.8 nm), zeta potential (−33.5 ± 2.6 mV), maximum EE (81.23 ± 1.1%) and DL (31.5 ± 2.9%). X-ray diffractometry spectra confirmed the encapsulation of ATR into NPs, whereas Transmission electron microscopy revealed that NPs were spherical in shape. Additionally, the ATR/SPI-WPC NPs significantly reduced cholesterol level as compare to SPI and WPC, individually. The results of internalization and MTT assay revealed efficient cellular uptake and cytocompatibility of the formulation. The results confirmed that SPI and WPC based bioactive material can be considered as promising biomaterial for encapsulation and enhancement of the therapeutic efficiency of ATR.

---

1. Introduction

According to WHO reports in 2015, obesity and cardiovascular disease (CVD) are the major causes of premature death worldwide annually. An estimated 17.5 million people died from CVDs in 2012, representing 31% of all global deaths.¹ The numbers of heart patients suffering from CVD worldwide is gradually increasing.² The patients associated with irregular levels of lipid profile, e.g. serum cholesterol (SC), serum triglycerides (ST), low density lipoprotein (LDL), high density lipoprotein (HDL), very low density lipoprotein (VLDL), will have a high risk of experiencing cardiovascular disorders³ and also contribute to develop secondary ailment, such as diabetes mellitus, obesity, hypothyroidism and renal insufficiency. The CVD related complications currently present a huge challenge for clinical practitioners to treat.⁴ Thus, a new drug delivery approach is required for the better management of hyperlipidemia.

Statins have been reported as cholesterol reducing drugs in several studies to reduce CVD mortality.⁵ Atorvastatin (ATR) is a well known drug which is frequently used to decrease the cholesterol level and prevent cardiovascular diseases. It is a potent competitive inhibitor of the HMG Co-A (3-hydroxy-3-methylglutaryl coenzyme A) enzyme involved in conversion of HMG Co-A to mevalonate in cholesterol biosynthesis in the liver.

Recent progress in protein based nanotechnology has shown significant potential for novel drug delivery systems.^6–8 Protein based NPs have unique properties including controlled particulate diameter, high surface to volume ratio, high loading capability, sustained release action and drug targeting potential.⁹ Proteins obtained from animal source (whey proteins, gelatin, casein) and from plant (soy proteins, pea proteins, cereal proteins) are widely used for encapsulation of active substances.¹⁰ These natural polymers present several advantages such as biodegradability, non-antigenicity, high nutritional value, easily modifiable surfaces, abundant renewable sources and astonishing binding capacity for various drugs as well as ease to scale up during manufacturing.^11–13 Moreover, they have excellent functional properties and also possess therapeutic properties, which make them interesting to process as drug carriers for various drugs.

Among the diverse range of protein based materials used for preparing NPs, soy protein isolate (SPI) and whey protein concentrate (WPC) have gained increasing popularity because of their unique ability of forming intra and intermolecular bonds, whereas the degree of crosslinking depends on the amine groups of the proteins.¹⁴ SPI is a commercial form of soy protein that contains more than 90% protein and 18 diverse amino acids.^15–17 It has been established that soy protein contributes to reduction of serum concentration of total cholesterol, low-density lipoproteins (LDLs), and triglycerides.^18,19 The presence of amino and carboxyl groups in SPI creates prominent site for crosslinking reaction. Several components associated with soy protein have been implicated in lowering cholesterol: trypsin inhibitors, saponins, isoflavons. Small amounts of the heat-stable Bowman–Birk inhibitor (trypsin inhibitors) may exert a hypo cholestremic effect by increasing the secretion of cholecystokinin. Saponins may contribute to cholesterol lowering by increasing bile excretion. Soy protein containing isoflavones are known to lower cholesterol significantly and also to inhibit formation of atherosclerotic lesions in primates. On the other hand, whey proteins work by hampering the absorption of cholesterol by way of reduced micellar cholesterol solubility in intestine.²⁰ Whey protein is a valuable by-product of the dairy industry^21,22 and has also been reported to hold cholesterol reducing activity,²³ which could be favorable for cardio vascular diseases. WPC consists of protein content of <80% (ref. 24) and is rich in hydrophilic groups such as –OH, –CONH–, –CONH₂, –COOH, and –SO₃H.^23,25 Genipin (Gn) is obtained from fruits of Gardenia jasminoides.²⁶ It is particularly advantageous in terms of being natural and a non toxic crosslinker with efficient property for the crosslinking of amino groups. Crosslinking process is also believed to improve the cholesterol reducing property of proteins (SPI and WPC).

It has been reported that SPI and WPC are promising biopolymers for the preparation of NPs by desolvation method.^27,28 Desolvation is a beneficial method to obtain stable nanoparticles with narrow size distribution. The type of desolvating agent (DA), the pH of the protein solution, the rate of the organic solvent added, stirring rate of the protein solution during the desolvation process are all important parameters to be considered²⁹ during optimization. Central composite design (CCD) was selected as the statistical based design of choice for the optimization of NPs. The novelty of the work is the use of Gn crosslinked SPI and WPC as an encapsulating material for the preparation of NPs with synergistic effect of biomaterial in lowering of cholesterol level in combination with ATR.^30,31

The objective of our study was to design and develop a new delivery approach for ATR by synthesizing the cross/SPI-WPC using non toxic amine–amine crosslinker (Gn). Further, cross/SPI-WPC would be used as a bioactive material to encapsulate ATR by desolvation method. The in vivo antihyperlipidemic activity of the optimized formulation was evaluated in rat model. Cellular uptake and MTT assay was performed in J774 cell lines to evaluate the biocompatibility and internalization of NPs within the cells.

2. Material and methods

2.1. Materials

ATR was received as a gift sample from Sun Pharmaceutical Industries Ltd., (India). Soy protein isolate and whey protein concentrate was purchased from Adeesh Agro Foods, (Haryana, India). Trinitrobenzene sulfonic acid (TNBS) reagent and dialysis membrane (molecular weight cut off 12 000–14 000) were purchased from Sigma–Aldrich. Gn was purchased from Challenge Bioproducts (Taichung, Taiwan). Triton X-100 was purchased from Himedia Laboratories Pvt. Ltd, (Mumbai, India). Membrane filters (0.45 μm) were purchased from Pall Life Sciences, India. Diagnostic kit for determination of lipid profile was obtained from Span Diagnostics Ltd, Gujarat, India. All other chemicals were of analytical grade and were purchased from Himedia, Mumbai and used as obtained.

2.2. Synthesis of SPI-WPC conjugates

Cross/SPI-WPC was synthesized by Gn fixed crosslinking of –NH₂ groups in proteins.³⁰ SPI (5% w/v) and WPC (5% w/v) were dissolved in double distilled water (1 h). The resulting solution was immediately crosslinked by dropwise addition of aqueous Gn solutions (0.5, 1, 1.5, 2% w/w respectively) and stirred for 1 h. The excess of crosslinker was removed by frequent washing with distilled water and then purified by ultra centrifugation for 20 min at 18 000 × g rpm (REMI CPR-24, Mumbai, India). The purified conjugates were lyophilized (Christ Alpha 1–4 lyophilizator, Osterode, Germany) and stored for further characterization.

2.3. Characterization of SPI-WPC conjugates

2.3.1. Degree of crosslinking. The TNBS assay was used to determine degree of crosslinking as illustrated by Bubnis et al.³¹ Weighed amount (5 mg) of cross/SPI-WPC was mixed with TNBS solution (0.5% w/v, 1 ml). The resultant solution was mixed with 1 ml of 4% w/v sodium hydrogen carbonate to make the system alkaline (pH approx 8.5) and heated on a water bath at 40 °C for 2 h. Afterwards, 12.24 N HCl (2 ml) was mixed with the solution and again heated at 70 °C for another 2 h. The absorbance of the solution was determined using UV-Vis Spectrophotometer (Labtronics LT-2910) after appropriate dilution at 415 nm. The degree of crosslinking was calculated by comparison of cross/SPI-WPC absorbance with pm/SPI-WPC absorbance (eqn (1)). The experiment was performed in triplicate and values reported in appendix Table 1.^25,32

Table 1 Summary of experimental design with their responses (particle size, zeta potential, EE, DL)^a

NPs code	Factors with levels		Responses
	X1 (w/v)	X2 (ml)	Particle size^b (nm)	Zeta potential (mV)	EE^b (%)	DL^b (%)
a X1 = cross/SPI-WPC w/v%, X2 = volume of DA (ml).b Average ± standard deviation.
ATR/SPI-WPC NPs-1	1	4	153 ± 1.24	−24.1	82.4 ± 2.33	54 ± 2.6
ATR/SPI-WPC NPs-2	3	4	157 ± 2.11	−39.4	85 ± 1.32	28 ± 3.1
ATR/SPI-WPC NPs-3	1	6	228 ± 0.89	−30.5	73 ± 1.78	48.6 ± 2.2
ATR/SPI-WPC NPs-4	3	6	272 ± 3.12	−38.9	79 ± 2.54	22 ± 1.7
ATR/SPI-WPC NPs-5	2	4	155 ± 1.76	−26.4	84 ± 2.11	34 ± 1.4
ATR/SPI-WPC NPs-6	2	6	248 ± 2.33	−33.5	76 ± 1.43	30 ± 2.8
ATR/SPI-WPC NPs-7	3	5	179 ± 1.82	−26.3	79 ± 1.1	21 ± 2.5
ATR/SPI-WPC NPs-8	1	5	169 ± 0.98	−21.4	78 ± 1.55	52 ± 1.9
ATR/SPI-WPC NPs-9	2	5	172 ± 2.12	−24	80 ± 2.5	40 ± 2.6
SPI-WPC NPs	3	6	343 ± 2.18	−11.1	—	—

---

2.3.2. Fourier transform infrared spectroscopy (FTIR). The FTIR spectra of the SPI, WPC, cross/SPI-WPC and lyophilized ATR/SPI-WPC nanoparticles were recorded using Shimadzu FTIR spectrophotometer. The measurements were carried out in the spectral regions of 4000–400 cm⁻¹ with a scanning frequency of 45 times and resolution of 4 cm⁻¹.

2.3.3. MS/MS analysis. The MS analyses of crosslinked material was done to detect whether Gn was involved in crosslinking chemically or physically, through Waters UPLC-TQD Mass spectrometer with Negative mode ESI-MS. The parameters used during analysis were capillary voltage 2700 V, sample cone voltage: 30.0 V, extraction cone voltage: 0.5 V, source temperature: 80 °C, desolvation temperature 150 °C, flow rate 0.5 μl min⁻¹, ion energy 2.0 V, collision energy 7.0 V.

For the MS analysis samples were prepared as per mentioned details. STD-Gn, pure form of Gn was dissolved in 5 ml HPLC grade methanol, R-Gn, cross/SPI-WPC was hydrolyzed in methanolic HCl at 60 °C for 1 h and supernatant obtained after centrifugation was evaporated to dryness. The residue was dissolved in 5 ml HPLC grade methanol and subjected to MS analyses, ME-Gn, cross/SPI-WPC was heated in HPLC grade methanol at 60 °C for 1 h and supernatant methanol was evaporated to dryness. The residue was redissolved in HPLC grade methanol and subjected to MS analyses.³³

2.4. Fabrication of ATR/SPI-WPC nanoparticles

ATR/SPI-WPC nanoparticles were prepared by desolvation method with slight modification using ethanol as DA.^26,27 In brief, cross/SPI-WPC was dissolved in double distilled water and stirred for 1 h. The pH of the solution was made alkaline (pH 9.0) using 2 M NaOH and kept overnight in a refrigerator. Desolvation of the solution was initiated by the addition of ethanol containing ATR at the rate of 1 ml min⁻¹ with sonication (Labsonic®-M, Sartorious stedium) at 50% amplitude for 10 min. The turbidity in the solution revealed formation of nanoparticles. The nanoparticulate suspension was centrifuged at 18 000 rpm (REMI CPR-24, Mumbai) for 10 min and pellets obtained were then washed, filtered (0.45 μm syringe filter), lyophilized (Christ Alpha 1–4 lyophilizator, Osterode, Germany) and stored at 4 °C for further studies.

2.5. Designs of experiment

There are several important factors which influence the physicochemical properties of the prepared NPs. Two factor, three level central composite design was developed to investigate the influence of independent variables such as amount of cross/SPI-WPC (%, w/v) and volume of DA (ml) at three different levels. Particle size (Y₁), zeta potential (Y₂), entrapment efficiency (EE, Y₃) and drug loading (DL, Y₄) were selected as response variables shown in Table 1. As per the CCD generated by software (Design Expert, Trial Version 8.0.7.1, Stat-Ease Inc., MN) nine experiments were conducted.

The second order polynomial model (eqn (2)) was used to establish the mathematical relationship between the independent variables.

Y = b₀ + b₁X₁ + b₂X₂ + b₃X₁X₂ + b₄X₁² + b₅X₂² (2)

where, Y is the response; b₀ is the intercept, and b₁, b₂, b₃, b₄ and b₅ are regression coefficients. X₁ and X₂ are individual effects; X₁² and X₂² are quadratic effects; X₁X₂ is the interaction effect.

2.6. Characterization of ATR/SPI-WPC nanoparticles

2.6.1. Particle size, polydispersity index and zeta potential. The mean particle size, polydispersity index (PDI) and zeta potential of ATR/SPI-WPC NPs were determined using laser light scattering at 657 nm and fixed angle of 90° Zetasizer NS 3000 (Malvern Instruments, UK). Zeta potential was determined using a disposable zeta cuvette. All experiments were performed at 25 °C. The data obtained after three measurements and average values with standard deviation were reported.

2.6.2. Nanoparticle recovery, EE and DL. Nanoparticle recovery (%) was calculated using eqn (3) after accurately weighing dried NPs

The ultracentrifugation method was used to determine EE and DL of ATR/SPI-WPC NPs suspension. For this purpose, specified suspensions were centrifuged (REMI CPR-24, Mumbai, India) for 20 min at 15 000 rpm and 4 °C to remove the free drug. The free drug in supernatant was diluted and absorbance taken using UV-VIS Spectrophotometer (Labtronics LT-2910) at 246 nm. All the experiments were performed in triplicate and mean values reported. The EE and DL was determined by the following equation.

2.6.3. X-ray diffractometry (XRD). X-ray diffractometer (PANalytical, X'Pert Data PW3050/60) was used to obtain wide angle XRD pattern of the samples ATR, SPI, WPC, pm/SPI-WPC, cross/SPI-WPC and lyophilized ATR/SPI-WPC nanoparticles. Powdered samples were placed in a glass sample holder. Cu-Kα radiation (k = 1.54 Å) in the 2θ range of 10–50° at 45 kV, 40 mA was used for all measurements at room temperature.

2.6.4. Scanning electron microscope (SEM) and transmission electron microscope (TEM) study. Morphological characteristics of the SPI, WPC and cross/SPI-WPC were observed by scanning electron microscope (SEM, Jeol Model JSM 6400, Tokyo). The samples were fixed to aluminum stubs with double-sided carbon adhesive tape and coating of dried samples was done by gold splutter coater under an argon atmosphere. Finally, coated samples were mounted on stub and observed. TEM was used to observe the surface morphology of SPI-WPC NPs and ATR/SPI-WPC NPs. Each sample was prepared by fixing small drop of the diluted suspension (25 μl) on carbon coated copper grids and allowed to air dry. The examination of specimen was carried out by transmission electron microscope (TECNAI 200 kV, Electron Optics) and images recorded for study.

2.6.5. In vitro release profile. The in vitro release studies were performed using dialysis bag method. ATR marketed formulation and optimized ATR/SPI-WPC NPs-10 were loaded into respective dialysis bags. The sink condition was maintained by adding sodium lauryl sulfate (0.1% w/v) to increase the solubility of poorly soluble ATR in dissolution media. The loaded bag was placed in release medium (200 ml) contained in a beaker incubated in a water bath maintained at 37 ± 1 °C and the media was kept under magnetic stirring at 100 rpm. 2 ml of samples were withdrawn at regular time interval with proper replacement of fresh medium. Drug content determination was carried out spectrophotometrically (Labtronics LT-2910) at 246 nm, in triplicate.

2.6.6. Stability of nanoparticles. The lyophilized ATR/SPI-WPC nanoparticles were subjected to stability studies, by evaluating particle size, PDI, zeta potential and drug content. The formulation was stored at 30 °C ± 2 °C and 65% RH ± 5% RH over a period of 180 days. The samples were withdrawn on 10^th, 90^th and 180^th day to determine the changes in formulations during storage.

2.6.7. Cell viability. J774 cells, a murine macrophage cell line (ATCC), was used to evaluate the cell viability by using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay.³⁴ J774 cells can be used to study cholesterol flux between cells and serum, which in turn is directly related to the cardiovascular disorders (increased cholesterol plaques in arteries). The cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum. The cells were incubated in 5% CO₂–95% (v/v) air atmosphere at 37 °C.

MTT assay was carried out by seeding the cells (5000 cells per well) in 96 well plate and incubated at 37 °C for 24 h. After incubation, cells were exposed to different concentrations of ATR, SPI-WPC NPs and ATR/SPI-WPC NPs (6.25, 12.5, 25, 50 and 100 μg ml⁻¹) dispersed in culture media for 24 h and 48 h followed by washing with HBSS (Hank's balanced salt solution). Thereafter, cells were incubated with 20 μl MTT for another 3 h. The purple formazan crystals were dissolved using DMSO. The absorbance of each well was measured at 570 nm through a microplate reader (Alere Microplate reader, AM, 2100).

2.6.8. Cellular uptake studies. Confocal laser scanning microscopy was used to study the localization of NPs in J774 cells. FITC loaded NPs suspension (200 μg ml⁻¹) were incubated with J774 cells (37 °C ± 2 °C with 5% CO₂ atmosphere) for 24 h followed by washing with PBS and fixation with paraformaldehyde (4% v/v) for 30 min. Cells were mounted over glass slide after appropriate washing and images captured using a confocal laser scanning microscope (Zeiss LSM510 Meta) at excitation wavelength of 490 nm and emission wavelength of 528 nm for FITC.

2.6.9. In vivo antihyperlipidemic study &estimation of HMG-CoA reductase activity. Albino Wistar rats weighing between 150–200 g were used to study antihyperlipidemic activity. The in vivo experiments were performed as per approval granted by Institutional Animal Ethics Committee (IAEC of Babu Banarasi Das Northern India Institute of Technology, Lucknow, protocol approval number BBDNIIT/IAEC/058/2014) as per Government of India norms. The animals were kept in well ventilated animal house in large polypropylene cages (22 °C ± 2 °C) maintained under photoperiodic condition. Animals were fed standard rat chow and water ad libitum. Care was taken that all guidelines are followed and a humane approach is followed. Animals were fasted for 18 h and then divided into eight groups (n = 6). Single dose of Triton X-100 (100 mg kg⁻¹) was administered by intraperitoneal (ip) injection to induce hyperlipidemia in rats. After 72 h of triton injection, animals received defined daily dose of standard drug, polymer and formulations for 7 days, after inducing hyperlipidemia.

Group 1 – control, carboxy methyl cellulose, (CMC: 0.5% w/v, p.o.).

Group 2 – hyperlipidemic control, triton X-100 (100 mg kg⁻¹, p.o.).

Group 3 – positive control, ATR (10 mg per kg per day p.o.) + triton X-100.

Group 4 – SPI (10 mg per kg per day p.o.) + triton X-100.

Group 5 – WPC (10 mg per kg per day p.o.) + triton X-100.

Group 6 – SPI + WPC + ATR (10 mg per kg per day p.o.) + triton X-100.

Group 7 – SPI-WPC NPs (10 mg per kg per day p.o.) + triton X-100.

Group 8 – ATR/SPI-WPC NPs-10 (10 mg per kg per day p.o.) + triton X-100.

Blood samples were collected in tubes by retro orbital route, under mild ether anesthesia on 8^th day and were centrifuged (2400 × g for 10 min) to separate serum. The extracted serum was assayed for ST, SC, HDL, LDL and VLDL by using commercial kits (Span Diagnostics Ltd, Gujarat, India).

HMG-CoA reductase activity was determined by method recommend by Venugopala Rao and Ramakrishnan.³⁵ Fresh liver homogenate, 0.5 ml, (10% tissue homogenate was prepared by using arsenate solution 1 g L⁻¹) was mixed with 0.5 ml of perchloric acid (50 ml L⁻¹). Samples were centrifuged (2000 rpm, 10 min) after 5 minutes. The obtained solution was filtered and 1 ml filtrate was added to 0.5 ml of freshly prepared hydroxyl amine reagent (2 mol L⁻¹ reagent; pH 5.5 for HMG-CoA and 2 mol L⁻¹ reagent; pH 2.1 for mevalonate). After 5 minutes, 1.5 ml of ferric chloride reagent was added and shaken well for 10 min. Absorbance was taken at 540 nm against blank (without homogenate) using double beam UV-Visible spectrophotometer (Labtronics LT-2910).

2.6.10. Histopathological study. At the end of the in vivo study, animals were sacrificed under mild anesthesia and liver was isolated for histopathology. The isolated liver was preserved in 10% buffered formalin solution. Tissues were processed as per routine histological procedure like washing and dehydration. Sectioning was done by semi-automated rotatory microtome. Hematoxylin and eosin were used for staining and morphological changes were observed under light microscope. The changes in the experimental histopathological parameters for liver tissues were graded as follows: (0) showing no changes, and (1), (2) and (3) indicating mild, moderate and severe changes, respectively.

2.7. Statistical analysis

All experiments were conducted three times and results expressed as a mean ± standard deviation. The cell viability and in vivo study were analyzed by one-way analysis of variance (ANOVA). The difference between the means was assessed using Dunnett's test using GraphPad Prism 5. p < 0.05, p < 0.01 was considered as statistically significant.

3. Result and discussion

3.1. Synthesis of SPI-WPC conjugate

SPI and WPC are obtained from natural sources and possess ample free amine groups representing ideal material for crosslinking and are also known to be cholesterol lowering proteins.^36,37 On the other hand, Gn is a non toxic crosslinker which is involved either, inter or intra-molecularly in crosslinking between free amine groups of proteins. Furthermore, it was thought worthwhile to consider ATR/SPI-WPC NPs as appropriate drug delivery systems for enhanced therapeutic efficiency of ATR with negligible toxicity.

3.2. Characterization of SPI-WPC conjugates

3.2.1. Degree of crosslinking. The results of crosslinking degree (CD) are presented in appendix Table 1. The data revealed that Gn is a good natural crosslinking agent due to its ability of efficient crosslinking of SPI with WPC containing amine groups. TNBS assay confirmed that CD of SPI with WPC ranged from 56.35 ± 0.6% to 66.72 ± 0.43% and 71.91 ± 0.38% as increasing Gn concentration from 0.5% w/w to 1.0% w/w and 1.5% w/w, respectively. Furthermore, the Gn concentration less than 0.5% w/w resulted in insufficient crosslinking and variation in PDI and particle size during preparation of trial batches (data not shown). On the other hand, Gn concentration increased from 1.5% w/w to 2.0% w/w and CD decreased to 66.91 ± 0.87% suggesting complete crosslinking of amine groups present in SPI and WPC at 1.5% w/w. Hence, Gn concentration (1.5% w/w) was selected for crosslinking of SPI with WPC. The appearance of cyan color after crosslinking demonstrated the reaction of Gn with amine groups of proteins. The ring opening reaction of Gn dihydropyran ring started with the nucleophilic attack of the amino group, followed by radical reaction of two amino-attached open rings.^30,38,39

3.2.2. Fourier transform infrared spectroscopy (FTIR). The crosslinking reaction was characterized by FTIR peaks. Fig. 1A shows peaks at 1660 cm⁻¹ (amine I), 1538 cm⁻¹ (amide II), 1238 cm⁻¹ (amide III) of SPI, while in Fig. 1B peaks at 1653 cm⁻¹ (amine I), 1528 cm⁻¹ (amide II), 1239 cm⁻¹ (amide III) corresponding to WPC are observed. Displacement of SPI and WPC peaks to cross/SPI-WPC (1638 cm⁻¹, 1384 cm⁻¹ and 1215 cm⁻¹) suggested that crosslinking reaction took place between amino groups of proteins as shown in Fig. 1C. The successful incorporation of ATR in NPs has been confirmed by the absence of ATR peaks at 3365 cm⁻¹ (N–H stretching), and 1651 cm⁻¹ (C=O stretching) respectively in FTIR peaks (Fig. 1D and E) of NPs.^26,40

Fig. 1 FTIR spectra of (A) SPI, (B) WPC, (C) cross/SPI-WPC, (D) ATR, (E) ATR/SPI-WPC nanoparticles.

3.2.3. MS/MS analysis. Appendix Fig. 1A presented the mass spectrum of STD-Gn: m/z (% abundance) 225 (M⁺ − 1, 100%), 207 (5%), 123 (25%), 101 (3%). The mass spectrum of the residue which was hydrolyzed with methanolic HCl of the cross/SPI-WPC produced the following major ion fragmentation besides other contamination peaks. The mass spectrum of R-Gn showed (Appendix Fig. 1B) major peaks of Gn: m/z 225 (M⁺ − 1, 40%), 207 (2%), 123 (12%), 101 (1%). On the other hand, mass pattern of ME-Gn did not show major peaks at m/z 207, 101 (Appendix Fig. 1C) which was observed in mass spectrum of STD-Gn. These results of MS analysis indicate that Gn is chemically involved in crosslinking reaction between amine groups of SPI and WPC as per previously reported results.^33,41,42

3.3. Fabrication of ATR/SPI-WPC nanoparticles

ATR/SPI-WPCNPs were prepared by desolvation process due to robustness and reproducibility for the preparation of stable and size controllable NPs and also to allow surface functionalization.^42,43 The addition of DA to the protein solution resulted in folding of protein initiated by the expulsion of water from the hydrophobic core. Furthermore, continuous addition of DA leads to nanoprecipitation of cross/SPI-WPC attributed to loss of water from the hydrophobic core resulting in folding of proteins and encapsulation of drug. The yield of nanoparticles was found to be high ranging from 66 ± 1.84% to 87 ± 3.11%.

3.4. Designs of experiment

ATR/SPI-WPC NPs preparation is based on desolvation method and the critical factors influencing the desolvation process were investigated by conducting nine experiments. The design of experiment with their observed response values is presented in Table 1. The model fitting was carried out using the values of investigated responses by applying one-way ANOVA (p < 0.05) for statistical evaluation and regression coefficient of the model equation was calculated (Table 2). Multiple regression analysis was used for model simplification through exclusion of non-significant terms (p > 0.05) from model equations.

Table 2 The results of ANOVA analysis for particle size (Y₁), zeta potential (Y₂), EE (Y₃) and DL (Y₄)

Response	Source	Sum of squares	df^a	Mean square	F value	p-Value (Prob > F)	R^2
a Is degree of freedom, S is significant at p > 0.05.
Y1	Model	19.45	5	3.89	213.62	0.0005 (S)	0.9972
	Cor total	19.50	8
Y2	Model	2.62	4	0.65	10.86	0.0201 (S)	0.9157
	Cor total	2.86	8
Y3	Model	0.34	2	0.17	32.29	0.0006 (S)	0.9150
	Cor total	0.37	8
Y4	Model	8.46	5	1.69	12.78	0.0309 (S)	0.9552
	Cor total	8.85	8

---

3.5. Optimization of conditions for preparation ATR/SPI-WPC NPs

The ATR/SPI-WPC NPs were characterized for particle size, zeta potential, EE and DL. The particles obtained were found in range from 153 ± 4.2 to 272 ± 1.5 nm. Amount of cross/SPI-WPC (% w/v) and volume of DA (ml) were the most important factors affecting particle size. The simplified model explains the relationship between the independent variables and the particle size presented in eqn (6).

Y₁ = +16.37X₁ + 3.13X₂ + 1.54X₁X₂ + 4.08X₁² + 0.24X₂² (6)

where X₁ and X₂ represents the amount of cross/SPI-WPC and volume of DA, respectively. The linear term of cross/SPI-WPC had a greater effect on the response variable (Y₁, particle size). The positive coefficient of X₁ indicated that higher level of cross/SPI-WPC increases the particle size. When the small amount of DA added to the protein solution it was found that molecules of cross/SPI-WPC remained suspended in the aqueous phase with no particles formation. While, the concentration of DA increased upto 40–60% v/v of protein concentration, the particle sizes increased progressively till it reached a stable value. However, further addition of DA (above then 60% v/v) caused no change in particle size. It is suggested that high DA content favors the exposure and interaction of the hydrophobic chains in protein molecules, which leads to formation of aggregates. The increase in average size at high ethanol concentrations has been reported in previous literature.^43,44 Further investigation focused on the effect of amount of cross/SPI-WPC on the particle size. Higher amount of cross/SPI-WPC resulted in the collision and aggregation of protein molecules, which in turn increases particle size.⁴⁵ The polydispersity index of all NPs was found to be below 0.5 (0.214–0.460). Appendix Fig. 3 showed particle size distribution of SPI, WPC, SPI-WPC NPs and ATR/SPI-WPC NPs.

Zeta potential is an important electrokinetic parameter which furnishes information regarding stability of NPs. The adjusted model for zeta potential is presented in eqn (7).

Y₂ = +5.12X₁ − 1.75X₂ + 0.82X₁X₂ + 0.19X₂² (7)

According to eqn (7), it is clear that linear term of cross/SPI-WPC amount (X₁) significantly affects the response variable. The positive coefficient of X₁ indicated that increase in cross/SPI-WPC increases the zeta potential. The cross/SPI-WPC is an encapsulating material for the preparation of NPs and it displayed a negative charge when prepared at high pH (9.0).²¹ In contrast, increase in DA volume weakened the electrostatic repulsion between protein molecules which resulted in protein aggregate and prevented their dissociation.²⁶

The simplified linear model which demonstrated the relationship between the independent variables and % EE is presented by eqn (8).

Y₃ = +0.90X₁ − 0.11X₂ (8)

It is observed from equation that positive coefficient of X₁ revealed that an increasing amount of cross/SPI-WPC lead to increase in % EE. The eqn (9) presents the modified quadratic model which demonstrates the relationship between the independent variables and DL. The linear term of cross/SPI-WPC had a positive effect on the response variable (Y₄, DL). High cross/SPI-WPC concentration resulted in small inter-particles spaces or crowded protein strands which could increase resistance to drug diffusion into the dispersing phase, leading to the incorporation of greater amount of ATR inside NPs. Another possible reason for high entrapment and drug loading is that ATR is a high molecular weight drug (1209.38 Da) and this attribute can be credited for slower diffusion of drug molecules from the polymeric matrix of particles.

Y₄ = +38.47X₁ − 0.56X₂ + 0.28X₁X₂ + 13.83X₁² − 0.026X₂² (9)

3.6. Validation

Optimization and validation of the experimental model was done in order to check the reliability of applied design. Response surface methodology (RSM) is a well established statistical approach which was applied to investigate the effect of independent variables on responses via 3D plots (plots shown in Appendix Fig. 2). The predicted model was generated by applying the criteria of minimum particle size and maximum zeta potential, EE and DL. The optimum level of cross/SPI-WPC concentration and volume of DA were determined to be 2.10% w/v and 4 ml respectively. At these levels, predicted responses of Y₁ (particle size), Y₂ (zeta potential), Y₃ (EE) and Y₄ (DL) were 154.36 nm, −30.26 mV, 83.67% and 35.46%, respectively. To validate the predicted model a new batch of NPs (ATR/SPI-WPC NPs-10) was prepared according to the optimized formula. The obtained response for the particle size, zeta potential, EE and DL were found to be 158.1 ± 3.8 nm, −33 ± 2.6 mV, 81.23 ± 1.1% and 31.5 ± 2.9% respectively. The comparison between particles and their predicted and obtained values indicated a good agreement with each other.

Fig. 2 XRD Pattern of (A) SPI, (B) WPC, (C) pm/SPI-WPC, (D) cross/SPI-WPC, (E) ATR, (F) ATR/SPI-WPC NPs-10.

Fig. 3 SEM images of (A) SPI, (B) WPC, (C) cross/SPI-WPC and TEM images of (D) SPI WPC NPs, (E) ATR/SPI WPC NPs-10.

3.7. Characterization of the optimal ATR/SPI-WPC nanoparticles

3.7.1. X-ray diffractometry (XRD). XRD patterns of ATR, SPI, WPC, pm/SPI-WPC, cross/SPI-WPC and lyophilized ATR/SPI-WPC NPs-10 are presented in Fig. 2. The less intense peak of SPI, WPC, pm/SPI-WPC, cross/SPI-WPC did not show any characteristic crystalline peaks and there was no change in physical state of cross/SPI-WPC due to crosslinking. The crystallographic character of ATR was confirmed by diffraction peaks (Fig. 2E) at about 10.45, 11.74, 12.06, 16.93, 19.37, 21.51, 22.60, 23.18 and 23.63 at 2θ with different signal intensities.⁴⁶ The XRD pattern of ATR/SPI-WPC NPs-10 (Fig. 2F) revealed the absence of diffraction peak corresponding to crystalline ATR (Fig. 2E) indicating that ATR is no longer present in a crystalline form when converted into nanoparticles and exists in the amorphous state.

3.7.2. SEM and TEM studies. The SEM micrograph of SPI and WPC revealed highly folded structure with rough surface. The formation of cross/SPI-WPC resulted in a particulate structure with smooth surface morphology due to addition of genipin, which increased the association between polypeptide chains of SPI and WPC. The TEM images of the SPI-WPC NPs (Fig. 2B) and ATR/SPI WPC NPs-10 (Fig. 2C) indicate that both type of NPs are spherical, fairly smooth, and with uniform surface. The appearance and shape of the NPs was not affected due to the same preparation method i.e. desolvation using ethanol as desolvating agent. The folding of proteins initiated upon addition of desolvating agent further led to nanoprecipitation cross/SPI-WPC to form NPs.

3.7.3. In vitro release profile. In vitro release of ATR from prepared ATR/SPI-WPC NPs-10 was evaluated using dialysis bag diffusion technique after incubation in phosphate buffer pH 7.4. The in vitro drug release from nanoparticles showed sustained release over 48 h with no considerable burst release (Fig. 4). This phenomenon may be attributed to an almost complete loading of ATR in NPs which leads to absence of unbound drug on the surface of the NPs. The cumulative drug released from ATR/SPI-WPC NPs-10 in 24 h was found to be 72.90 ± 1.72%, while the complete release (approximately 100%) of drug was observed in case of ATR after 24 h 90.80 ± 2.0% of the drug was released within 48 h, after which, a slowdown was observed at 60 h (91.19 ± 2.0%) which was almost constant upto 72 h (90.98 ± 2.4%). This sustained release pattern of drug from NPs may be due to the Gn induced crosslinked SPI and WPC as a material for the preparation of NPs which possibly prevented quick dissolution of crosslinked polymer matrix. In addition, at neutral pH 7.4, WPC was found to be more stable. Thus, resistance offered by NPs against proteolytic degradation and water uptake could be attributed to the presence of whey protein which delayed the expulsion of drug from the crosslinked matrix. The results obtained were found to be in accordance with previously published studies.⁴⁴

Fig. 4 In vitro release profile of ATR in phosphate buffer pH 7.4 from ATR/SPI-WPC NPs-10 and ATR solution.

3.7.4. Stability of nanoparticles. The lyophilized ATR/SPI-WPC NPs-10 exhibited no considerable difference in particle size, PDI and drug content as represented in Table 3. Zeta potential of the NPs was found to alter from −44.5 to −45.5 after 180 days of storage. The stability result suggested that NPs were stable upto 180 days at 25 °C.

Table 3 Particle size, PDI and drug loading of ATR/SPI-WPC NPs-10 recorded over a period of 180 days, storage at room temperature

Time (days)	Particle size (nm)	PDI	Drug loading (%)
0	158.1 ± 11.3	0.239 ± 2.0	31.5 ± 2.3
10	162 ± 14.1	0.265 ± 2.4	30.6 ± 1.6
90	177 ± 10.7	0.281 ± 2.2	27.3 ± 2.6
180	181.6 ± 15.2	0.283 ± 1.3	25.7 ± 3.9

---

3.7.5. Cell viability. A cell viability study was performed in order to evaluate the cytocompatibility of the prepared SPI-WPC NPs and ATR/SPI-WPC NPs with J774 cells using MTT assay at incubation period of 24 h and 48 h. This was investigated by testing the cell viabilities of J774 cells for 24 h and 48 h. The cell viabilities were found to be more than 80% for SPI-WPC NPs (90.34 ± 2.0%) and ATR/SPI-WPC NPs (80.12 ± 2.30%) in comparison to ATR (50.20 ± 4.0) upto a dose of 100 μg ml⁻¹ after incubation period of 24 h (Fig. 5A). On the other hand, after incubation period of 48 h the cell viability of ATR/SPI-WPC NPs was to decrease with the increasing dose of NPs, although the cell viability was found to be higher (68 ± 3.80%) in comparison to ATR (39.30 ± 1.5%) at the highest dose of 100 μg ml⁻¹ (Fig. 5B).

Fig. 5 MTT assay for percentage cell viability of ATR, SPI-WPC NPs and ATR/SPI-WPC NPs after (A) 24 h and (B) 48 h incubation period with increasing concentration of NPs, data are expressed as mean ± SD of three independent experiments. * denotes a statistically significant (p < 0.05) difference from the control group. Confocal microscopic images of control (C–F) and FITC labeled ATR/SPI-WPC NPs (G–J) showing the cellular uptake of nanosized NPs in J774 cells. (C and G) Bright field, (D and H) green fluorescent channel, (E and I) blue channel, (F and J) overlay of channels.

Conclusively, cell viability studies suggested that prepared NPs appeared to affect cell viability less than ATR. The NPs were non-toxic, probably due to the use of naturally occurring dietary proteins (SPI, WPC) as well as natural crosslinker (Gn). SPI, WPC and Gn proved to be non toxic^47–49 and thus it could be assumed that prepared NPs would also be biocompatible. The combination of SPI and WPC is inert and acts as single component system. Furthermore, the NPs can be regarded as safe drug delivery carriers for future applications.

3.7.6. Cellular uptake study. Confocal microscopy images (Fig. 3C–J) of florescent ATR/SPI-WPC NPs displayed the cellular uptake potential of prepared delivery system in murine macrophages J774 cell lines. The cellular uptake of particles is influenced by size of particles, charge and incubation period with respective cells. Confocal microscopy revealed intracellular uptake of the optimized NPs in J774 cells. Many cellular uptake pathways have been described for internalization of nanoparticles into cells. The transport mechanisms include active transport, passive transport, caveolae and clathrin-mediated endocytosis or pinocytosis. However some reports suggest that active transport, passive transport, caveolae and clathrin-mediated endocytosis or pinocytosis mechanisms are responsible for the nanoparticles made up of proteinaceous material (fibroin and gelatin).^30,44 Based on reports, we can hypothesize that protein based nanocarrier systems could internalize as a result of one or a combination of above said uptake mechanisms.

The majority of NPs co-localized in the cell cytoplasm and not in the nucleus of the cell, which was revealed by strong green fluorescent channel as shown in Fig. 5H. Nano-sized (below 250 nm) and negatively charged NPs are appropriate for cellular uptake and these features ensure better interaction with cationic site of cell surface. Furthermore, this can occur due to an increased retention time of NPs at the cell surface which increases the probability of particle internalization.^50,51

3.7.7. In vivo antihyperlipidemic study &estimation of HMG-CoA reductase activity. The present study was performed to compare the anticholesterol potential of ATR/SPI-WPC NPs with SPI, WPC, SPI-WPC, SPI + WPC + ATR in hypercholestrimic rats. Fig. 6 demonstrated that triton administered rats displayed significantly (p < 0.05 and p < 0.01) increased level of SC, ST, LDL and VLDL and decreased level of serum HDL (p < 0.05 and p < 0.01) in comparison to control group. The obtained data showed that SPI and WPC were effective in preventing the increase cholesterol level in rats. The SPI + WPC + ATR groups showed better anticholesterol activity as compared to SPI and WPC group attributed to synergistic effect of soy, whey and drug.

Fig. 6 Serum lipids levels & H/M ratio in liver extract, Con: rats + CMC (0.5% w/v), triton: rats + triton, ATR: rats + triton + ATR (10 mg kg⁻¹), SPI: rats + triton + SPI, WPC: rats + triton + WPC, SPI + WPC + ATR: rats + triton + SPI + WPC + ATR, ATR/SPI-WPC NPs: rats + triton + ATR/SPI-WPC NPs, SPI-WPC NPs: rats + triton + SPI-WPC NPs data are expressed as means ± SEM, n = 6. *p < 0.05 and **p < 0.01 represent significant differences when compared with the Control group. ^#p < 0.05 and ^##p < 0.01 represent significant differences when compared with the Triton group. SC: serum cholesterol, ST: serum triglyceride, HDL: high density lipoproteins, LDL: low density lipoproteins, VLDL: very low density lipoproteins, AI: anthrogenic index = ST/HDL, H/M ratio: HMG-CoA/Mevalonate ratio.

The highlight of this work is that ATR/SPI-WPC NPs group displayed lower level of lipids (SC = 62.5 ± 3.1, ST = 56.7 ± 3.8, LDL = 16.2 ± 4.6, VLDL = 12.8 ± 0.95) as compared to ATR group (SC = 89.67±, ST = 65.67 ± 4.10, LDL = 25.6 ± 3.1, VLDL = 13 ± 0.9) and triton treated group (SC = 125 ± 3.2, ST = 110 ± 3.2, LDL = 75 ± 2.9, VLDL = 17.6 ± 1.9). Therefore, it was concluded that ATR/SPI-WPC NPs showed potential hypolipidemic effect. A possible explanation for this might be the synergistic effect of SPI, WPC and ATR, as several studies reported that soy protein^18–20 and whey protein²³ holds cholesterol reducing activity which could be favorable for cardio vascular diseases. The mechanisms of the lipid-lowering effects of soy protein are due to the presence of several components although the whey protein works by hampering the absorption of cholesterol through reduced cholesterol solubility in lipid micelles.²³ Antherogenic index (AI) can be calculated by total cholesterol (SC)/total high density lipoprotein (HDL), and is an indicator of cardiovascular diseases. A significant (p < 0.05 and p < 0.01) reduction in AI ratios of ATR/SPI-WPC NPs (1.46 ± 0.69) was found when compared with triton administered group (4.46 ± 0.76). Hence, it can be concluded that optimized ATR/SPI-WPC NPs would provide better protection against cardiovascular diseases.

The degree of cholesterol synthesis was determined by H/M assay based on quantifying the activity of HMG-CoA reductase enzyme. The H/M ratio for control, triton, ATR, SPI, WPC, SPI + WPC + ATR, ATR/SPI-WPC NPs and SPI-WPC NPs was found to be 3.41, 2.52, 3.30, 2.8, 2.5, 3.04, 3.7 and 2.5, respectively. The higher H/M ratio indicates a lower extent of cholesterol synthesis, which was observed in the ATR/SPI-WPC NPs treated group. In contrast triton administered group displayed a higher cholesterol synthesis (2.52) compared to control group (3.41). Thus, our results suggest that developed nanoparticulate system can be used to lower the risk of cardiovascular heart disease.

3.7.8. Histopathological study. The changes in the experimental histopathologic parameters for liver tissues were graded as follows: (0) showing no changes and (1), (2) and (3) indicating mild, moderate and severe changes, respectively. The observed histopathological changes in hepatic tissue are shown in Fig. 7 and histopathology changes in liver tissues are also summarized in Table 4. Microscopic observations (Fig. 7A) as well as the data (Table 4) revealed that liver from rats (control group) demonstrated no change in Kupffer cells (K; score = 0), sinusoidal spaces (S; score = 0) with normal hepatocytes (H; score = 0). Fig. 7B shows histopathology of liver in toxic group, characterized by large degree of tissue damage such as necrosis of hepatocytes (score = 2), sinusoidal dilatation (score = 3), activated Kupffer cells (score = 3). The liver tissue of rats treated with ATR displayed regular hepatic structure with dark stained nucleus in Fig. 7C and a mean score of 1.5. Rats treated with SPI-WPC NPs (Fig. 7D) showed normal hepatic morphology attributed to anticholesterol activity as well as non toxic property of SPI and WPC and a mean score of 2. The liver sections (Fig. 7E) of rats treated with ATR/SPI-WPC NPs-10 showed absence of sinusoidal dilatations (score = 1), necrosis in hepatocytes (score = 0), with darkly stained nucleus activated Kupffer cells (score = 1) and preserved parenchymal structures as a proof of hepatic structure recovery. The smallest mean score value (ATR/SPI-WPC NPs-10; mean score = 0.75) was attributed to high recovery of hepatic structure.

Fig. 7 Histological evaluation of the liver stained by H & E stain (A) rats + CMC (0.5% w/v) (40×); (B) rats + Triton (40×); (C) rats + Triton + ATR (40×); (D) rats + Triton + SPI-WPC NPs (40×); (E) rats + Triton + ATR/SPI-WPC NPs-10 (40×).

Table 4 Scores of liver histopathology for all groups^a

Histopathology scores of observation for each group	Control	Toxic	ATR	SPI-WPC NPs	ATR/SPI-WPC NPs-10
a Where (0) indicates no changes and (1), (2) and (3) indicate mild, moderate and severe changes respectively.
Activated Kupffer cells	0	3	2	2	1
Sinusoidal dilatation	0	3	2	2	1
Cytoplasmic vacuolation	0	2	1	2	1
Hepatocytes	0	2	1	2	0
Mean score	0	2.5	1.5	2	0.75

---

4. Conclusions

Recent reports show frequent use of biomaterial based excipients for encapsulation of various drugs. It is considered to be a green approach to utilize entities already present in nature, or their modified forms, than to load the environment with new chemical entities. In the present study, we synthesized a novel biomaterial by genipin induced crosslinking of SPI and WPC and reported the physicochemical characteristics by FTIR, degree of crosslinking, MS/MS spectroscopy. The NPs were successfully prepared using novel biomaterial and loaded with ATR by desolvation method. The characterization and optimization of NPs was done by central composite statistical design using particle size, zeta potential, EE and DL. The results indicated that optimized NPs (ATR/SPI-WPC NPs-10) showed sustained release of ATR over a period of 72 h. The findings of cell viability studies suggest that using SPI, WPC, Gn makes the delivery system non-toxic and biocompatible. From the in vivo and histological characterization, it may be concluded that ATR loaded in novel biomaterial is a promising delivery system which proved to have better anticholesterol activity as compared to that of pure drug. However, further evaluation of these carriers can be performed for their potential to treat hypercholesterolemia associated diseases such as diabetes, obesity, hypothyroidism and Gaucher disease, as future scope.

Conflict of interest

The authors confirm that content of this article has no conflict of interest.

Acknowledgements

The kind support of Sophisticated Analytical Instrument Facility (SAIF), Panjab University, Chandigarh, in XRD and TEM studies is acknowledged.

References

1. World Health Organization-WHO, Cardiovascular diseases (CVDs), Fact sheet N°, Geneva, 2015, p. 317.
2. D. Kumar, V. Parcha, A. Maithani and I. Dhulia, Effect and evaluation of antihyperlipidemic activity guided isolated fraction from total methanol extract of Salvador aoleoides (Decne.) in Triton WR-1339 Induced hyperlipidemic rat, Pharmacogn. Mag., 2012, 8, 314–318.
3. M. E. Shahira, A. S. Essam, M. H. Fathalla and A. G. Salah, Antihyperglycemic and antihyperlipidemic effects of the methanol extracts of Cleome ramosissima Parl., Barleriabispinosa (Forssk.) Vahl. and Tribulusmacropterus Boiss, Bull. Fac. Pharm., 2014, 52, 1–7.
4. P. K. Biswal, N. R. Pani and P. K. Dixit, Effects of carbohydrate polymers in self-microemulsified tablets on the bioavailability of atorvastatin: In vitro–in vivo study, Life Sci., 2015, 135, 92–100.
5. F. L. Pereira, V. B. Oliveira, C. T. R. Viana, P. P. Campos, M. A. N. Silva and M. G. L. Brandao, Antihyperlipidemic and antihyperglycemic effects of the Brazilian salsaparrilhas Smilax brasiliensisS preng. (Smilacaceae) and Herreriasal saparrilha Mart. (Agavaceae) in mice treated with a high-refined-carbohydrate containing diet, Food Res. Int., 2015, 76, 366–372.
6. M. Kim, S. Jin, J. Kim, H. J. Park, H. S. Song, R. H. Neubert and S. J. Hwang, Preparation, characterization and in vivo evaluation of amorphous atorvastatin calcium nanoparticles using supercritical antisolvent (SAS) process, Eur. J. Pharm. Biopharm., 2008, 69, 454–465.
7. V. P. Torchilin, Nanotechnology in Drugs, Imperial College Press, London, 2nd edn, 2008.
8. S. K. Sahoo and V. Labhasetwar, Nanotech approaches to drug delivery and imaging, Drug Discovery Today, 2008, 8, 1112–1120.
9. A. O. Elzoghby, W. M. Samy and N. A. Elgindy, Protein-based nanocarriers as promising drug and gene delivery systems, J. Controlled Release, 2012, 161, 38–49.
10. A. Nesterenkoa, I. Alric, F. Silvestre and V. Durrieu, Vegetable proteins in microencapsulation: A review of recent interventions and their effectiveness, Ind. Crops Prod., 2013, 42, 469–479.
11. J. A. Jenkins, H. Breiteneder and E. N. Mills, Evolutionary distance from human homologs reflects allergenicity of animal food proteins, J. Allergy Clin. Immunol., 2007, 120, 1399–1405.
12. H. Li, K. Zhu, H. Zhou and W. Peng, Effects of high hydrostatic pressure treatment on allergenicity and structural properties of soybean protein isolate for infant formula, Food Chem., 2012, 132, 808–814.
13. L. Chen, G. E. Remondetto and M. Subirade, Food protein-based materials as nutraceutical delivery systems, Trends Food Sci. Technol., 2006, 17, 272–283.
14. F. Hammann and M. Schmid, Determination and Quantification of Molecular Interactions in Protein Films: A Review, Materials, 2014, 7, 7975–7996.
15. P. Kumar, K. P. Sandeep, S. Alavi, V. D. Truong and R. E. Gorga, Preparation and characterization of bio-nanocomposite films based on soy protein isolate and montmorillonite using melt extrusion, J. Food Eng., 2010, 100, 480–489.
16. L. Xiang, C. Tang, J. Cao, C. Wang, K. Wang, Q. Zhang, Q. Fu and S. G. Zhao, Preparation and Characterization of Soy Protein Isolate (Spi)/Montmorillonite (mmt) Bionanocomposites, Chin. J. Polym. Sci., 2009, 27, 843–849.
17. R. Kumar, V. Choudhary, S. Mishra, I. K. Varma and B. Mattiason, Adhesives and plastics based on soy protein products, Ind. Crops Prod., 2001, 16, 155–172.
18. W. W. Wong, E. O. B. Smith, J. E. Stuff, D. L. Hachey, W. C. Heird and H. J. Pownell, Cholesterol-lowering effect of soy protein in normocholesterolemic and hypercholesterolemic men, Am. J. Clin. Nutr., 1998, 68, 1385–1389.
19. R. M. Arliss and C. A. Biermann, Do soy isoflavones lower cholesterol, inhibit atherosclerosis and play a role in cancer prevention?, Holistic Nursing Practice, 2002, 16, 40–48.
20. J. W. Erdman, Soy Protein and Cardiovascular Disease, A Statement for Healthcare, Professionals From the Nutrition Committee of the AHA, Circulation, 2000, 14, 2555–2559.
21. S. Gunasekaran, S. Ko and L. Xiao, Use of whey proteins for encapsulation and controlled delivery applications, J. Food Eng., 2007, 83, 31–40.
22. A. Abbasi, Z. Emam-Djomeh, M. A. E. Mousavi and D. Davoodi, Stability of vitamin D3 encapsulated in nanoparticles of whey protein isolate, Food Chem., 2014, 143, 379–383.
23. K. Marshall, Therapeutic Applications of Whey Protein, Altern. Med. Rev., 2004, 9, 136–156.
24. H. Winkler, W. Vorwerg and M. Schmid, Synthesis of hydrophobic whey protein isolate by acylation with fatty acids, Eur. Polym. J., 2015, 62, 10–18.
25. P. Aramwit, T. Siritientong, S. Kanokpanont and T. Srichana, Formulation and characterization of silk sericin–PVA scaffold crosslinked with genipin, Int. J. Biol. Macromol., 2010, 47, 668–675.
26. Z. Teng, Y. Luo and Q. Wang, Nanoparticles Synthesized from Soy Protein: Preparation, Characterization and Application for Nutraceutical Encapsulation, J. Agric. Food Chem., 2012, 60, 2712–2720.
27. I. Gulseren, Y. Fang and M. Corredig, Zinc incorporation capacity of whey protein nanoparticles prepared with desolvation with ethanol, Food Chem., 2012, 135, 770–774.
28. S. R. Kaup, N. Arunkumar, L. K. Bernhardt, R. G. Vasavi, S. S. Shetty, S. R. Pai and B. Arun Kumar, Antihyperlipedemic activity of Cynodondactylon extract in high-cholesterol diet fed Wistar rats, Genomic Med., Biomarkers, Health Sci., 2011, 3, 98–102.
29. M. R. Law, N. J. Wald and A. R. Rudnicka, Quantifying effect of statins on low density lipoprotein cholesterol, ischaemic heart disease, and stroke: systematic review and meta-analysis, BMJ, 2003, 326, 1423–1427.
30. R. You, Y. Xu, G. Liu, Y. Liu, X. Li and M. Li, Regulating the degradation rate of silk fibroin films through changing the genipin crosslinking degree, Polym. Degrad. Stab., 2014, 109, 226–232.
31. W. A. Bubnis and C. M. Ofner, Anal. Biochem., 1992, 207, 129–133.
32. R. N. Kale and A. N. Bajaj, Ultraviolet Spectrophotometric Method for Determination of Gelatin Crosslinking in the Presence of Amino Groups, J. Young Pharm., 2010, 2, 90–94.
33. R. Meena, K. Prasad and A. K. Siddhanta, Development of a stable hydrogel network based on agar–kappa-carrageenan blend cross-linked with genipin, Food Hydrocolloids, 2009, 23, 497–509.
34. J. Kanoujia, M. Singh, P. Singh and S. A. Saraf, Novel genipin crosslinked atorvastatin loaded sericin nanoparticles for their enhanced antihyperlipidemic activity, Mater. Sci. Eng., C, 2016, 69, 967–976.
35. A. Venugopala Rao and S. Ramakrishnan, Indirect Assessment Hydroxymethylglutaryl Co-A Reductase (NADPH) Activity in Liver Tissue, Clin. Chem., 1975, 21, 1523–1525.
36. C. E. West, C. J. K. Spaaij, W. M. Clous, H. P. Twisk, M. P. Goertz, R. W. Hunnard, M. W. Kuyvenhoven, R. Vander meer, W. F. Roszkowaski and A. Sanchez, Comparison of the hypocholesterolemic effects of dietary soybean protein with those of formaldehyde-treated casein in rabbits, J. Nutr., 1989, 119, 843–856.
37. A. C. Beynen, C. E. West, C. J. K. Spaaij, J. Huisman, P. Van Leeuwen, J. B. Schutte and W. H. Hackeng, Cholesterol metabolism, digestion rates and postprandial changes in serum of swine fed purified diets containing either casein or soybean protein, J. Nutr., 1990, 120, 422–430.
38. Q. Li, X. Wang, X. Lou, H. Yuan, H. Tu, B. Li and Y. Zhang, Genipin-crosslinked electrospun chitosan nanofibers: Determination of crosslinking conditions and evaluation of cytocompatibility, Carbohydr. Polym., 2015, 130, 166–174.
39. F. I. Mi, S. S. Shyu and C. K. Peng, Characterization of ring-opening polymerization of genipin and pH-dependent cross-linking reactions between chitosan and genipin, J. Polym. Sci., Part A: Polym. Chem., 2005, 43, 1985–2000.
40. Z. Teng, Y. Luo and Q. Wang, Carboxymethyl chitosan–soy protein complex nanoparticles for the encapsulation and controlled release of vitamin D3, Food Chem., 2013, 141, 524–532.
41. R. Meena, K. Prasad and A. K. Siddhanta, Effect of genipin, a naturally occurring crosslinker on the properties of kappa-carrageenan, Int. J. Biol. Macromol., 2007, 41, 94–101.
42. M. Wackera, A. Zensi, J. Kufleitner, A. Ruff, J. Schutz, T. Stockburger, T. Marstaller and V. Vogel, A toolbox for the upscaling of ethanolic human serum albumin (HSA) desolvation, Int. J. Pharm., 2011, 414, 225–232.
43. X. H. Li, Y. Li, Y. F. Hua, A. Y. Qiu, C. Yang and S. Cui, Effect of concentration, ionic strength and freeze-drying on the heat-induced aggregation of soy proteins, Food Chem., 2007, 104, 1410–1417.
44. Y. W. Won and Y. H. Kim, Recombinant human gelatin nanoparticles as a protein drug carrier, J. Controlled Release, 2008, 127, 154–161.
45. G. M. Kavanagh, A. H. Clark and S. B. Ross-Murphy, Heat induced gelation of globular proteins: part 3. Molecular studies on low pH beta-lactoglobulin gels, Int. J. Biol. Macromol., 2000, 28, 41–50.
46. A. Bathool, G. D. Vishakante, M. S. Khan and H. G. Shiva kumar, Development and characterization of atorvastatin calcium loaded chitosan nanoparticles for sustain drug delivery, Adv. Mater. Lett., 2012, 3, 466–470.
47. J. Zhang, L. Liang, Z. Tian, L. Chen and M. Subirade, Preparation and in vitro evaluation of calcium-induced soy protein isolate nanoparticles and their formation mechanism study, Food Chem., 2012, 133, 390–399.
48. M. Mekhaila, K. Jahan and M. Tabrizian, Genipin-crosslinked chitosan/poly-l-lysine gels promote fibroblast adhesion and proliferation, Carbohydr. Polym., 2014, 108, 91–98.
49. M. L. Cross and H. S. Gill, Modulation of immune function by a modified bovine whey protein concentrate, Immunol. Cell Biol., 1999, 77, 345–350.
50. S. Patil, A. Sandberg, E. Heckert and W. Self, Protein adsorption and cellular uptake of cerium oxide nanoparticles as a function of zetapotential, Biomaterials, 2007, 28, 4600–4607.
51. M. G. Mekhail, A. O. Kamel, G. A. S. Awad and N. D. Mortada, Anticancer effect of atorvastatin nanostructured polymeric micelles based on stearyl-grafted chitosan, Int. J. Biol. Macromol., 2012, 51, 351–363.

---

Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c6ra16830b

---