Preparation and characterisation of atorvastatin and curcumin‐loaded chitosan nanoformulations for oral delivery in atherosclerosis

Abstract

Atorvastatin known to be a potential inhibitor of HMG‐CoA reductase involved in the synthesis of cholesterol. It is touted as miracle drug due to its profound effect in decreasing the low‐density lipoproteins in blood. Unfortunately, the high dosage used poses side‐effects relatively in comparison to other statins. On the other hand, curcumin has a diverse therapeutic potential in health and disease. However, the poor aqueous solubility and low bioavailability hinders the therapeutic potential of it when administrated orally. Therefore, it was thought to minimise the frequency of atorvastatin doses to avoid the possibility of drug resistance and also to overcome the limitations of curcumin for desirable therapeutic effects by using nanocarriers in drug delivery. In this investigation, synergistic effect of atorvastatin and curcumin nanocarriers was encapsulated by chitosan polymer. The chitosan nanocarriers prepared by ionic gelation method were characterised for their particle size, zeta potential, and other parameters. The drug‐loaded nanocarriers exhibited good encapsulation efficiency (74.25%) and showed a slow and sustained release of atorvastatin and curcumin 60.36 and 61.44%, respectively, in a span of 48 h. The drug‐loaded nanocarriers found to be haemocompatible and qualified for drug delivery in atherosclerosis.

1 Introduction

Atherosclerosis is the leading causes of death all over the world. Hyperlipidemia is known to be the leading risk factor for atherosclerosis. Myocardial and cerebral infraction is the main clinical syndromes resulting from atherosclerosis [1, 2]. The HMG‐CoA reductase inhibitors (statins) are widely used in medical practice which includes anti‐inflammatory and immunomodulatory properties and plaque stabilisation in atherosclerosis [3]. Atorvastatin, an orally administered drug is a member of drug class known as statin. Clinically it is being used for the treatment of elevated total cholesterol (TC), low density lipoprotein and triglycerides (TGs), and high density lipoprotein cholesterol [4, 5]. Atorvastatin stabilises plaque and reduced the risk of stroke by 16%, through anti‐inflammatory and other mechanisms. The oral absorption rates of all the drug candidates of statin group exhibits different. Atorvastatin oral absorption is reported by 30% and of which 12% would be the bioavailability [6]. Due to the cause for the poor bioavailability atorvastatin is absorbed primarily from the intestine and to a lesser extent from the stomach and presystemic metabolism. Researchers have shown that saturable transport system involves in the hepatic uptake of atorvastatin in rats in vivo [7]. Metabolic studies in animals reported that >98% of the dose of atorvastatin was recovered in the faeces within 48 h and it is metabolised extensively by cytochrome P‐450 3A4 in the gut wall and liver [8]. Curcumin has a diverse therapeutic potential in health and disease. It is evident from investigations that due to free radical scavenging property, the potential molecule exhibits anti‐inflammatory, anti‐proliferative, anti‐cancer, anti‐angiogenic, anti‐diabetic, anti‐malaria and anti‐cardiovascular diseases activity [9, 10]. Recently, in vivo tests have revealed that curcumin could inhibit the development of atherosclerosis in the apoE/LDLR‐double knockout mice fed with Western diet [11]. In addition, long‐term curcumin treatment lowers plasma and hepatic cholesterol and suppresses early atherosclerotic lesions comparable with the protective effects of lovastatin. At the molecular level, the anti‐atherogenic effect of curcumin is known to mediate via multiple mechanisms including altered lipid, cholesterol and immune gene expression [12, 13].

In one supporting study, curcumin has increased faecal excretion of bile acids and cholesterol, both in normal and hypercholesteremic rats. This promising biliary drainage is a plausible explanation for the reduction of tissue cholesterol on curcumin feeding [14]. Further, curcumin diet also significantly decreased serum TG by 27%, TC by 33.8%, and LDL‐cholesterol by 56%, respectively, as compared with control group. The curcumin‐supplemented diet also significantly lowered the atherogenic index by 48% as compared with control group [15, 16].

In recent days, investigators are shifting their attention towards the use of natural bioactive compounds such as curcumin to load into the nanocarriers for targeted delivery applications [17]. Today, non‐toxic, biocompatible and biodegradable nanoparticles with various colloidal dimensions are being developed to enhance the penetration ability, reduce the frequency of doses, toxicity and to improve the therapeutic efficacy [18, 19, 20]. A number of clinical and experimental studies have demonstrated the effect of atorvastatin on atrial fibrillation, but the results are equivocal [21]. Due to technological innovations, today it is possible to enhance the bioavailability and improve the efficacy and safety of atrial fibrillation by developing the biodegradable combination of atorvastatin and curcumin‐loaded chitosan nanoparticles for oral delivery. Chitosan has one such ability to increase membrane permeability, both in vitro and in vivo. Chitosan has the potential of serving as an absorption enhancer across intestinal epithelial for its mucoadhesive and permeability enhancing property [22]. Chitosan microparticles and nanoparticles made by chemical cross‐linking are not preferred owing to their physiological toxicity. Chitosan is polycationic and can interact with negatively charged species such as tripolyphosphate [23]. Several methods used, an ionic gelation (IG) method developed by Calvo et al. has been found to be the suitable system to prepare stable and biocompatible chitosan nanoparticles [24]. In the present study, we have attempted to prepare biodegradable chitosan nanoparticles with and without drugs (atorvastatin and curcumin). Drug‐loaded chitosan nanoparticles [atorvastatin and curcumin nanocarriers (ACCNS)] are prepared with atorvastatin and curcumin either free‐form or in combination by IG technique. Blank nanoparticles (BCNS) are also prepared to compare and contrast with the drug‐loaded chitosan nanoparticles. Such nanoparticles are characterised for physicochemical properties including in vitro release and evaluated for their hemocompatibility.

2 Materials and methods

2.1 Materials

Chitosan (low molecular weight) was purchased from Sigma (St. Louis, MO, USA), Pentasodium tripolyphosphate (TPP) was purchased from Sigma (St. Louis, MO, USA), Atorvastatin calcium salt procured from Indswift Laboratory (Mumbai, India), Curcumin was purchased from Sigma (St. Louis, MO, USA), Tween 80 and Acetic acid was purchased from Hi‐media Chemical Co. (Mumbai, India). Double distilled water was used throughout the study. All other reagents are all of analytical grade.

2.2 Preparation of atorvastatin and curcumin combined drugs loaded chitosan nanoparticles

Chitosan nanoparticles were produced based on an IG method of TPP and chitosan reported by Calvo et al. with slight modification [25]. Nanoparticles were spontaneously obtained upon mixing the aqueous 0.05% (w/v) TPP solution (5 ml) with acidic 0.1% (w/v) chitosan solution (10 ml) using ultrasonication (50% Duty Cycle, 40 amp for 6 min). ACCNS were formed by the addition of atorvastatin dissolved in methanol (1.5 mg/ml), curcumin dissolved in ethanol (3.5 mg/ml) and 2% w/v tween 80 to chitosan solution and followed by the TPP solution in a drop‐wise manner. ACCNS formed were concentrated by centrifugation (Beckman T 20 rotor, USA) at 16,000 g in a 10 μl glycerol bed for 30 min. The supernatants were discarded, and resultant nanoparticles suspended in water and lyophilised (Labconco, Kansas City, MO, USA). In order to examine the effect of concentration of reacting molecules on particle size and encapsulation efficiency (EE), a range of chitosan (0.100, 0.200 and 0.300% (w/v)) and TPP (0.05% and 0.100% (w/v)) were used. BCNS were prepared in the same way by omitting the drugs.

2.3 Physicochemical characterisation of nanoparticles

2.3.1 Size measurement, zeta potential and morphological characterisation of nanoparticles

The nanoparticles prepared were subjected for assessment to find out the mean particle size, particle size distribution, polydispersity index and zeta potential by dynamic light scattering (DLS) with particle size analyser (Nano ZS, Malvern) at a wavelength of 635 nm and with a scattering angle of 90° at room temperature. In this process, we measured the diluted suspension of nanoparticles using appropriate volume of 0.1 mM NaCl and placing in the measurement cell. All measurements were performed in triplicate. An aliquot of freeze dried nanoparticles (2 mg) were used for shape and size analysis by scanning electron microscope (SEM) (JNCASR, Bangalore, India.). The observation was performed using a 20 kV, LaB6 (or tungsten filament) SEM equipped with an Everhart–Thornley secondary electron detector and a Cambridge four quadrant back scatter detector LEO 1530 (LEO 1455VP Cambridge, England) operated using 0.5 mA filament current. Sample was prepared as aqueous dispersion of nanoparticles were finely spread over a glass slide and dried under vacuum. The dried slide was placed onto carbon conductive double‐side tape (Euromedex, France) and dried further at room temperature. The processed nanoparticles were coated with gold (2 nm) and placed inside the vacuum column of the microscope after pumping the air out of the chamber.

2.3.2 Fourier transform infrared spectroscopy

The FTIR spectrum of the specimen was recorded with Nicolet IR 200 (Thermo Electron Corporation, USA). The spectral analysis was carried out in the wavelength region between 4000 and 400 cm⁻¹ at room temperature using KBr pellets (Merck, IR grade) for blank (BCNS) and drug‐loaded nanoparticles (ACCNS). The samples were allowed to form pellets at pressure of 10.3 × 10⁴ Pa.

2.3.3 Differential scanning calorimetry (DSC)

The thermal behaviour of BCNS, ACCNS, atorvastatin and curcumin was analysed using a Mettler‐Toledo 821^e DSC module controlled by STAR^e software (Mettler‐Toledo GmbH, Switzerland). Approximately ∼5 mg of freeze‐dried nanoparticles was analysed in aluminium pans, and heated from 30 and 360°C at a heating rate of 10°C/min per cycle. Inert atmosphere was maintained by purging nitrogen at the flow rate of 100 ml/min. The empty aluminium was used as the standard reference material to calibrate the temperature and energy scale of the DSC apparatus.

2.3.4 X‐ray diffractometry (XRD)

XRD was used to investigate the physical (crystalline or amorphous) form of atorvastatin and curcumin molecules dispersed within the chitosan matrix of the nanocarriers. The XRD patterns of BCNS, ACCNS, atorvastatin and curcumin were recorded on an X‐ray diffractometer (Siemen's, Kristallofex D‐5000 Munich, Germany) with Cu as anode material and crystal graphite monochromator, operated at a voltage of 40 Kv and a current of 30 mA. The samples were analysed in the 2θ angle range of 2° to 50° and the process parameters were set as follows: step size of 0.03° (2θ), scan step time of 0.5 s.

2.3.4 Encapsulation efficiency (%)

The drug content in the prepared ACCNS was calculated by the difference between the total amount of atorvastatin and curcumin added during the preparation and the amount of drug present in the supernatant after centrifugation. The atorvastatin and curcumin present in the supernatant was determined spectrophotometrically by reading the absorbance at 246 and 426 nm, separately (UV‐Vis Spectrophotometer Model UV1800, Shimadzu, Japan). Dilutions were made with phosphate buffer saline (PBS). All measurements are performed in triplicate. The EE of atorvastatin and curcumin of the nanoparticles are calculated as follows:

$$\mathrm{EE}(\text{\%})=\frac{\mathrm{Total}\mathrm{amount}\mathrm{of}\mathrm{the}\mathrm{drug}-\mathrm{free}\mathrm{drug}\mathrm{in}\mathrm{the}\mathrm{supernatant}}{\mathrm{Total}\mathrm{amount}\mathrm{of}\mathrm{drug}}\times 100$$

2.3.5 Drug release studies

Drug release studies were carried out by dialysis method. ACCNS were redispersed in freshly prepared in PBS (5 ml, pH 7.4) and taken into dialysis membrane bag with (12 kDa) after tying at both the ends. During dialysis, the bag was placed in a jar containing PBS (150 ml) and incubated at 37°C in a shaking water bath (50 rpm). The amount of atorvastatin and curcumin released from the atorvastatin and curcumin combination‐loaded chitosan nanoparticles was measured by sampling out 1 ml each time at predetermined time intervals (0.5, 1, 2, 3, 4, 6, 8, 10, 12, 16, 20, 24, 36, and 48 h). From an aliquot, the amount of atorvastatin and curcumin released was determined spectrophotometrically at 246 and 426 nm. A standard calibration curve was drawn using atorvastatin and curcumin as reference standard.

2.3.6 Hemocompatibility

Fresh blood was obtained from a healthy volunteer and collected in a blood collection tube containing anticoagulant (EDTA, 0.5 mg). The blood was centrifuged (2000 rpm, 20 min) at room temperature using REMI 24C centrifuge to separate the erythrocytes. The concentrated leukocyte band (a Buffy‐coat) and a small portion of the plasma was removed. Later, the concentrated RBCs in the packed cells were separately collected and washed thrice with normal saline (0.9% NaCl). The RBC cells and saline were taken in 1:1 ratio and centrifuged (2000 rpm, 10 min). The supernatant was discarded and washings were repeated thrice. Washed RBCs were further diluted to a 50% hematocrit by adding normal saline. Hemolysis experiments were followed in accordance with a method used previously in our laboratory with slight modifications.

100 µl cell suspensions taken into a clean dry test tube was added with an appropriate negative control in normotonic condition. The positive control was prepared with 100 µl cell suspensions by diluting with double distilled deionised water (3 ml) and the RBCs lysis was compared. Freeze‐dried BCNS, ACCNS and combination of atorvastatin and curcumin were redispersed and sonicated in a saline solution to give 0.2% suspensions. An aliquot of 100 µl of BCNS, ACCNS and combination of atorvastatin and curcumin suspension with different concentrations was added with 100 µl of RBC suspension and made to 3 ml by adding normal saline. The experiment was carried out in the triplicate. The mixture was incubated for 1 h at 37°C in a water bath (ILE instrument, Bangalore). The reaction was terminated using 50 µl of gluteraldehyde (2.5%). The samples were then centrifuged at 1000 rpm for 15 min and the absorbance of the supernatant was measured at 540 nm using UV‐VIS spectrophotometer (Model UV1800, Shimadzu, Japan).

$$\mathrm{Hemolysis}(\mathrm{\%})=\frac{\mathrm{Absorbance}\mathrm{of}\mathrm{the}\mathrm{sample}}{\mathrm{Absorbance}\mathrm{of}\mathrm{the}\mathrm{positive}\mathrm{control}}\times 100$$

3 Results and discussion

The chitosan/TPP ratio is critical and controls the size and the size distribution of the nanoparticles. The size characteristics have been found to affect the biological performance of chitosan/TPP nanoparticles. For this reason, we studied the effect of the chitosan/TPP ratio on the size characteristics of the nanoparticles to find the optimum ratio those results in nanoparticles with small size and a narrow size distribution. In doing so, we also compared and identified possible agreement of these results with those obtained using the theoretical approach to identify which chitosan/TPP ratio and what kind of ionic interactions give the optimum size for the nanoparticles.

The effects of two different variables (chitosan and TPP concentration) and ultrasonication (50% duty cycle at 40 amp for 6 min) on the particles size, size distribution, zeta potential, drug EE of chitosan nanoparticles. As the size matters in delivering the cargo in drug delivery systems, it necessitates the appropriate formulations by achieving the better drug and polymer ratio for potential therapeutic effects. The drug free chitosan nanoparticles (BCNS) were prepared considering two different variables namely polymer (chitosan) and cross‐linking agent (TPP) using ultrasonication (50% duty cycle at 40 amp for 6 min). The formulations were carried out by keeping one parameter variable and rest constant. The effect of TPP (0.05 and 0.1%), chitosan (10/20/30 mg) concentration and ultrasonication is an important parameter to reduce the size and polydispersity index.

The chitosan nanoparticles BCNS 1 to BCNS 6 were measured by DLS at 27°C, which exhibited differences in their size characteristics ranging between BCNS1 (73.16 ± 13.38 with PDI 0.210 ± 0.01 and zeta potential +30.1 ± 4.56) and BCNS6 (132.3 ± 1.735 with PDI 0.319 ± 0.083 and zeta potential +20.4 ± 2.29). The BCNS1 shows the size of the nanoparticles decreases as compared with BCNS2 to BCNS6 (Table 1). Polydispersity index shows narrow distribution and zeta potential is high due to the appropriate electrostatic interactions between positive charge of chitosan polymer chains and negative charge of drug molecules and TPP molecules. Even though formulation method used for BCNS1 was suitable for further drug loading, we still repeated every formulation (BCNS1 to BCNS6) for the preparation of drug loaded nanoparticles and characterised for size and zeta [26]. We then choose the best formulation which showed small sized nanoparticles with low PDI and high zeta for further characterisation and drug delivery.

Table 1.

Size, PDI and zeta potential drug free chitosan nanoparticles (BCNS)

Sl. no	Formulations code	Chitosan, mg	0.05% TPP solution, ml	Particle size, nm	PDI	Zeta potential
1	BCNS1	10	5	73.16 ± 13.38	0.210 ± 0.01	+30.1 ± 4.5
2	BCNS2	20	5	94.47 ± 9.67	0.273 ± 0.04	+26.6 ± 2.7
3	BCNS3	30	5	106.4 ± 3.11	0.257 ± 0.05	+24.8 ± 0.8

Sl. no	Formulations code	Chitosan, mg	0.1% TPP solution, ml	Particle size, nm	PDI	Zeta potential
1	BCNS4	10	4	110.6 ± 1.13	0.236 ± 0.09	+23.4 ± 1.0
2	BCNS5	20	4	119.2 ± 8.20	0.298 ± 0.08	+21.6 ± 0.2
3	BCNS6	30	4	132.3 ± 1.735	0.319 ± 0.083	+20.4 ± 2.2

Similarly the combination of atorvastatin and curcumin‐loaded chitosan nanoparticles consisted (ACCNS1 to ACCNS6) consisted 1.5 and 3.5 (atorvastatin and curcumin), 10/20/30 mg (chitosan) and 5 ml, 4 ml (0.05% and 0.1% TPP). The effect of chitosan concentration on drug‐loaded nanoparticles was studied, where smaller size nanoparticles were obtained when the lower concentration of polymer was used. The cross linking agent (TPP) and drug concentration also affects the particle size and zeta potential with respect to the chitosan concentration.

The chitosan nanoparticles ACCNS 1–ACCNS 6 were measured by DLS at 27°C, which exhibited differences in their size characteristics ranging between 105.3 ± 0.11 nm and 191.6 ± 1.10 nm with PDI of 0.229 ± 0.01 to 0.357 ± 0.09. However, the size of the nanoparticles began to increase on increasing the concentration of chitosan (0.1 to 0.3%) and TPP (0.05 to 0.1%). The results could be attributed that increased concentration of chitosan solution in the preparation system would certainly influence on the viscosity rise. While determining the effect of TPP concentration on the size of nanoparticles. Further, the addition of TPP influenced the size increase at 0.1% (191.6 ± 1.10 nm with 0.354 ± 0.12 PDI) and decreases 0.05% (105.3 ± 0.11 nm with 0.293 ± 0.06 PDI) concentration (Table 2). This increase in the particle size may be due to saturation of cationic groups (–NH⁺ ₃) on chitosan solution to the incoming anionic groups (P₃ O^–5 ₁₀) from TPP. From data, it was evident that chitosan nanoparticles could only be produced when used in a specific concentration range of chitosan and TPP. Any change in the concentration of TPP and chitosan might lead to either particle aggregation or no particle formation. Another important factor observed was that a decrease in TPP concentration leads to a narrower polydispersity distribution. The sonication energy also influence the smaller size and monomodal distribution profile as compared with higher concentration of TPP, which showed increase size and higher polydispersity index [27]. The small amount of surfactant also plays an important role in preparation process and in the protection of the droplets, because it can avoid the coalescence of globules. It was also observed that the size distribution became narrower. The obtained results shows that increase in chitosan concentration led to decrease of EE of atorvastatin and curcumin combination. It has been previously reported that the highly viscous nature of the gelation medium hinders the encapsulation of drugs [28]. Lower concentration of chitosan and TPP lead to better EE (74.25 ± 1.16 and 73.62 ± 1.44). Hence it was supposed that relatively lower viscosity of chitosan with lower concentration promotes the encapsulation of drugs and gelation between chitosan and TPP (Table 2) [29]. The charge of both chitosan and TPP solution has a great effect on the ionic interaction. The zeta potential is an important index for the stability of nanoparticles suspension. The zeta potential increased with the increasing of drug encapsulated, hence the stability of the drug increased with respect to the increased encapsulation. This may be due to the availability of free NH³⁺ groups on the polymer. The present study indicates decreased concentration of TPP and chitosan will increase the zeta potential. It is commonly used to characterise the surface charge property of the nanoparticles. The zeta potential reflects the electric potential of the particles and is influenced by the composition of the particle and the medium in which it is dispersed. The developed chitosan nanoparticles were in the range of +16.8 ± 1.15–+30.1 ± 4.56 mV, having been shown to be stable in suspension, as the surface charge prevents the aggregation of the particles. It can also be used to determine whether a charged active material is encapsulated within the centre of the nanocapsule or adsorbed on to the surface (Tables 1 and 2). This zeta potential indicates that chitosan nanoparticles were stable. The effect of ultrasonication, highest energy released during sonication causes a fast dispersion of the organic phase, leading to the formation of small nanodroplets of a monomodal distribution. These results show that sonication energy and time play an important role in the size and size distribution of the nanoparticles.

Table 2.

Size, PDI, encapsulation efficiency and zeta potential of the ACCNS

Sl. no	Formulations code	Atorvastatin + Curcumin, mg	Chitosan, mg	0.05% TPP solution, ml	Particle size, nm	PDI	Encapsulation efficiency, %	Zeta potential
1	ACCNS 1	1.5 + 3.5 = 5	10	5	105.3 ± 0.11	0.293 ± 0.06	74.25 ± 1.16	+30.1 ± 4.56
2	ACCNS 2	1.5 + 3.5 = 5	20	5	133.8 ± 1.05	0.257 ± 0.11	71.42 ± 1.36	+22.3 ± 0.61
3	ACCNS 3	1.5 + 3.5 = 5	30	5	174.9 ± 0.17	0.229 ± 0.01	69.64 ± 1.20	+22.0 ± 0.90

Sl. no	Formulations code	Atorvastatin + Curcumin, mg	Chitosan, mg	0.1% TPP solution, ml	Particle size, nm	PDI	Encapsulation efficiency, %	Zeta potential
1	ACCNS 4	1.5 + 3.5 = 5	10	4	110.4 ± 0.98	0.340 ± 0.10	73.62 ± 1.44	+21.0 ± 0.75
2	ACCNS 5	1.5 + 3.5 = 5	20	4	137.7 ± 2.40	0.357 ± 0.09	69.63 ± 1.34	+19.8 ± 1.33
3	ACCNS 6	1.5 + 3.5 = 5	30	4	191.6 ± 1.10	0.354 ± 0.12	67.42 ± 1.37	+16.8 ± 1.15

The morphology of the scanning electron microscopic analysis, it is clear that BCNS1 and ACCNS1 chitosan nanoparticles are more spherical shape, smoother surface and small size. This is an important observation because interaction of the nanoparticles is very dependent on shape of the nanoparticles. Spherical nanoparticles will not have much interaction, hence there circulation will be prolonged in the body (Fig. 1). However BCNS1 and ACCNS1 ratios were found suitable ratios for formation of chitosan nanoparticles. It is evident from the results shown in Tables 1 and 2. The chitosan and TPP concentration increases it results in the formation of larger size of the nanoparticles. This increase in size may be due to insufficient TPP for poor gelation of chitosan solution. Based on above observations ACCNS1 ratios were found to optimum for chitosan nanoparticles formation in terms of size, polydispersity index, zeta potential and EE. Fig. 1 shows the SEM images of BCNS1 and ACCNS1 chitosan nanoparticles.

Fig. 1.

SEM of

(a) Drug free chitosan nanoparticles (BCNS1) and (b) Atorvastatin and curcumin combination drug‐loaded chitosan nanoparticles (ACCNS1)

3.1 Fourier transform infrared spectroscopy

The FTIR spectrum showing percentage transmission (%T) versus wave number (cm⁻¹) of atorvastatin, curcumin, chitosan and atorvastatin and curcumin combination‐loaded chitosan nanoparticles have been shown in Fig. 2. The three characteristic peaks of OH, N‐H stretching and C=O stretching at 3424.01, 1648.38 and 1073.25 cm⁻¹, respectively. The comparison of chitosan and combined drugs loaded chitosan nanoparticles is different. However, chitosan nanoparticles exhibited similar peaks but with a negligible shift for OH, N‐H stretching and C=O stretching at 3429.43, 1636.64 and 1088.92 cm⁻¹. The chitosan and ACCNS peak 3424.01 and 3429.43 cm⁻¹ become wider, its indicating that hydrogen bonding is enhanced. The spectrum of FTIR consistent with the result of chitosan film modified by phosphate, and it could be attributed linkage between the phosphoric and ammonium ion [30]. From the dissolution point of view, the chitosan is related to the protonation of free amine groups and break down of strong intra‐ and intermolecular hydrogen bonding. It is evident from the figure that atorvastatin calcium and curcumin in nanoparticles does not undergo any chemical reaction with any of the excipients used in the preparation.

Fig. 2.

FTIR of

(a) Atorvastatin, (b) Curcumin, (c) Chitosan and (d) Atorvastatin and curcumin combination‐loaded chitosan nanoparticles

3.2 Differential scanning calorimetry

During nanoparticles preparation hydrophobic drugs should not be having any crystals inside the polymer matrix proper drug release. In DSC analysis, atorvastatin and curcumin showed a strong endothermic peak at 157.68°C and 180.27°C corresponding to the melting of drug, due to crystalline nature of drugs. The DSC profile of combined drugs loaded chitosan nanoparticles (ACCNS1) displayed broad but no peaks in the region (Fig. 3) accompanied by endothermic peaks is due to the Tg relaxation enthalpy of the chitosan. It confirms that amorphous or disordered‐crystalline phase of curcumin in the chitosan matrix. These results are in agreement with XRD analysis, that no crystal formation of atorvastatin and curcumin are present inside the nanoparticles that ultimately reflects homogeneous dispersion of drugs inside chitosan nanoparticles matrix [31].

Fig. 3.

DSC of

(a) Atorvastatin, (b) Curcumin, (c) Chitosan and (d) Atorvastatin and curcumin combination‐loaded chitosan nanoparticles

3.3 X‐ray diffractometry (XRD)

The Fig. 4 shows the XRD patterns of atorvastatin, curcumin, chitosan and atorvastatin and curcumin combination‐loaded chitosan nanoparticles. The high intensity peak present in pure drugs revealing that the drug is present in crystalline form. It is clear that the characteristic diffraction peaks were observed only in the case when the free drug is present. Whereas the characteristic peaks were not exhibited by chitosan nanoparticles. This indicates that no crystals of free drug are present inside the nanoparticles that ultimately reflect homogeneous dispersion of drug inside the nanoparticle matrix [32, 33, 34].

Fig. 4.

XRD of

(a) Atorvastatin, (b) Curcumin, (c) Chitosan and (d) Atorvastatin and curcumin combination‐loaded chitosan nanoparticles

3.4 Drug release studies

The in vitro release studies were carried out at physiological pH 7.4 and comparison of the release profile of pure atorvastatin and curcumin drugs with the release profile of drugs entrapped chitosan nanoparticles (ACCNS) showed slow and sustained release of combined drugs entrapped in the chitosan nanoparticles. Drug bioavailability increases due to slow and sustained drugs release of the chitosan nanoparticles and prolong the therapeutic effect [35]. The drug release of atorvastatin and curcumin from ACCNS 1 to ACCNS 6 chitosan nanoparticles exhibited burst release of atorvastatin (12.52 ± 1.2% to 15.39 ± 1.3%) and curcumin (14.47 ± 1.3% to 14.66 ± 1.4%) at first 30 min and later on showed a kind of biphasic release pattern with a gradual increase in releasing drug until 48 h period of atorvastatin (60.36 ± 1.2% to 68.48 ± 1.3%) and curcumin (61.44 ± 1.3 to 69.47 ± 1.3%) shown in Fig. 5. The initial burst release might be the result of rapid dissolution of the drugs crystals located at or present on the surface of the nanoparticles. The instantaneous release from the initial burst effect would allow the drug to gain an opportunity to enter into plasma in vivo to exert the pharmacological activity at the nanoscale level. 39.41 ± 1.2 drug release was observed in the in the first 12 h, later the drug was comparatively slower, which continued to release until 48 h. In the subsequent continued phase of release drug showed a kind of gradual retardation and the remaining releases accounted in the range atorvastatin (60.36 ± 1.2% to 68.48 ± 1.3%) and curcumin (61.44 ± 1.3 to 69.47 ± 1.3%). This was due to drug, polymer and TPP concentration in the chitosan nanoparticles. The increased concentration of the polymer increased the size of the particles. This would allow better diffusion of the dissolution medium in to the particles, otherwise which will slow down the dissolution due to the thickened wall of the particles wrapping around the polymer over and over again resulting in multi‐layered encapsulation. The atorvastatin and curcumin releases from the dialysis bag for all chitosan nanoparticles is evidently attributed to the prolong release function of chitosan nanoparticles. The ACCNS1 is much slower drug releases compare to other chitosan nanoparticles. It could be further considered as a slow and sustained, oral drug delivery system for atorvastatin and curcumin. It is very important for clinical applications.

Fig. 5.

In vitro Release of atorvastatin and curcumin from ACCNS 1 to ACCNS 6 nanoformulations

3.5 Hemocompatibility

The hemocompatibility study was conducted to ascertain the safety of the nanoparticles prepared using human RBCs. Hemocompatibility tests of chitosan nanoparticles ACCNS1 to ACCNS6 were carried out to assess the per cent hemolysis. In this test the samples were taken in three different concentrations 0.5, 1.5 and 1.5 mg to compare the degree of damage to the RBCs in comparison with double distilled water. The range of hemolysis of RBCs is 0.192 to 0.298% for ACCNS. In this compatibility study, all the six chitosan nanoparticles were found to be non‐toxic to the RBCs and could be considered for in vivo studies to further the investigations. The chitosan nanoparticles had appropriate amount of atorvastatin and curcumin (1.5 and 3.5 mg) and the drug released from the nanoparticles would not cause any hemolysis at that concentration [35]. In comparison, atorvastatin and curcumin combination‐loaded chitosan nanoformulations were found to be potential formulations for sustained release of drug and exhibited no adverse effects and found to be negligible hemolysis (Fig. 6).

Fig. 6.

Hemolysis (%) of atorvastatin and curcumin combination‐loaded chitosan nanoformulations

4 Conclusion

Atorvastatin and curcumin‐loaded chitosan nanocarriers can be prepared by cross linking of chitosan and tripolyphosphate. The chitosan nanoparticles were found to have excellent EE designated to oral delivery. FTIR showed that there is no interaction between the atorvastatin, curcumin while in combination as drugs and with the chitosan polymer, also. These DSC results, taken together, suggest that the encapsulation process produces a marked decrease in crystallinity of atorvastatin and curcumin confers to this drug a nearly amorphous state. XRD analysis also shows amorphous dispersion within the chitosan matrix of the chitosan nanoparticles. The in vitro release profile of atorvastatin and curcumin from chitosan nanoparticles showing initial burst release within 30 min and followed by slow and sustained release in an incremental form until 48 h. The hemolysis studies showed negligible amount of toxicity. From the characterisation studies, it is evident that the nanocarriers qualify themselves for the targeted drug delivery in chemotherapeutic applications of atherosclerotic diseases.