Claims:We claim,
1. A formulation of primaquine phosphate - chitosan nanoparticles, comprising:
a) primaquine phosphate;
b) chitosan; and
c) sodium tripolyphosphate;
wherein the amount of primaquine phosphate is 10 mg, chitosan is 0.1% and sodium tripolyphosphate is in the range of 0.24 to 0.74 mg/ml in the formulation of primaquine phosphate - chitosan nanoparticles.
2. The primaquine phosphate - chitosan nanoparticles as claimed in claim 1, wherein the nanoparticles are in the size range of 100 to 900 nm.
3. The primaquine phosphate - chitosan nanoparticles as claimed in claim 1, wherein the drug encapsulation efficiency of nanoparticles are in the range of 90-97%.
4. The primaquine phosphate - chitosan nanoparticles as claimed in claim 1, wherein the polydispersity index of nanoparticles are in the range of 0.188 to 0.362.
5. The primaquine phosphate - chitosan nanoparticles as claimed in claim 1, wherein zeta potential of nanoparticles are in the range of 17.2 to 34.2 mV.
6. A process for the preparation of primaquine phosphate - chitosan nanoparticles, comprising:
a) dissolving chitosan in an aqueous solution of acetic acid;
b) dissolving primaquine phosphate in chitosan solution of step a);
c) separately, dissolving sodium tripolyphosphate in distilled water;
d) adding sodium tripolyphosphate solution of step c) in chitosan primaquine phosphate solution of step b);
e) stirring solution of step d) using magnetic stirrer at 1000 rpm at room temperature for overnight;
f) recovering the nanoparticles by centrifugation of the formulation of step e) at 18,000 rpm and collecting supernatant.
Dated this 9th day of January, 2022
To be signed digitally by
(Sanchita Tewari)
Agent for the Applicant
Patent Agent (IN/PA 2711)
, Description:Technical Field of the Invention
The present invention relates to a primaquine phosphate loaded chitosan nanoparticles. The present invention also relates to a process for the preparation of primaquine phosphate loaded chitosan nanoparticles. Primaquine phosphate loaded chitosan nanoparticles of the present invention has sustained drug release profile for up to 24 hours.
Background of the Invention
Malaria is one of the deadly and life-threatening disease. It is caused by female anopheles mosquito parasites that infect people by biting and spreads infections. There were an estimated 229 million cases of malaria worldwide in 2019, and high death rate was estimated. Children under 5 years are the most susceptible age group affected by malaria and accounted for 67% of all malaria deaths worldwide. Malaria preventive drugs and strategies for controlling malaria are effective tools in prevention as well as in the treatment. Usage of antimalarial drugs are also very useful in malarial treatment (https://data.unicef.org/topic/child-health/malaria/). The main objective of the treatment of any disease is to effectively develop and delivered drug molecules to their respective target places. This objective is achieved only if drugs are developed and reached the target tissue efficiently enough so that they perform their respective actions properly. Prevention of drugs from digestive juices, instability, low absorption or degradation of drugs by enzymes are various problems that are associated with targeted delivery of medicine. To overcome medicine delivery related problems nanoparticles are designed which have specific characteristic properties useful for the delivering of required quantity of drugs. Nanoparticles are considered effective in drug delivery in respect of stability, uniform and small size, larger surface area with improved drug delivery properties. Polymers also shows better examples as a drug delivery carrier and helps in improved drug solubility, permeability and bioavailability. Desirable polymers have biodegradability, biocompatibility and nontoxicity, they bind with drug nanoparticles and shows improvement in drug bioavailability and solubility. Among polymers chitosan works as a good polymer for nanoparticles drug delivery.
Controlled release nanoparticles drug delivery system is an important step which can be achieved by using chitosan as a polymer with sodium tripolyphosphate (TPP). Chitosan and TPP at specific concentrations show best carrier characteristics in drug administration. In medicament delivery, developing improved drug carriers and formulation with desired qualities and effective against diseases are always promising (Gupta, N., Rajera, R., Nagpal, M. and Arora, S., 2013. Primaquine loaded chitosan nanoparticles for liver targeting. Pharmaceutical Nanotechnology, 1(1), pp.35-43). However, there is still a need for more effective and efficient drug delivery system with suitable carriers for the treatment of malaria.
Objects of the Invention
The main objective of the present invention is to provide a primaquine phosphate loaded chitosan nanoparticles.
Another object of the present invention is to provide a process for the preparation of primaquine phosphate loaded chitosan nanoparticles.
Yet another object of the present invention is to provide a formulation of primaquine phosphate loaded chitosan nanoparticles sustained drug release profile.
Another object of the invention is to provide a primaquine phosphate loaded chitosan nanoparticles useful for the treatment of malaria.
Summary of the Invention
Accordingly, the present invention provides a formulation of primaquine phosphate - chitosan nanoparticles, comprising of primaquine phosphate; chitosan; and sodium tripolyphosphate; wherein the amount of primaquine phosphate is 10 mg, chitosan is 0.1% and sodium tripolyphosphate is in the range of 0.24 mg/ml to 0.74 mg/ml.
In an embodiment of the invention, the primaquine phosphate loaded chitosan nanoparticles are in the size range of 100 to 900 nm.
In an embodiment of the invention, the polydispersity index (PDI) of primaquine phosphate loaded chitosan nanoparticles is in the range of 0.188 to 0.362.
In an embodiment of the invention, the zeta potential (ZP) of primaquine phosphate loaded chitosan nanoparticles is in the range of 17.2 to 34.2 mV.
The present invention also provides a process for the preparation of primaquine phosphate loaded chitosan nanoparticles, which comprises of dissolving chitosan in an aqueous solution of acetic acid; dissolving primaquine phosphate in chitosan solution; separately dissolving sodium tripolyphosphate in distilled water; adding sodium tripolyphosphate solution in chitosan primaquine phosphate solution above, stirring using magnetic stirrer at 1000 rpm at room temperature for overnight; recovering the nanoparticles by centrifugation of the formulation at 18,000 rpm and collecting supernatant.
Brief Description of drawings
In the drawings accompanying the specification, Figure 1 shows the encapsulation efficiency of 90%, 93% and 97% for nanoparticle formulations F1, F2 and F3.
In the drawings accompanying the specification, Figure 2 shows particle size distribution of nanoparticles.
In the drawings accompanying the specification, Figure 3 shows Zeta potential of nanoparticles.
In the drawings accompanying the specification, Figure 4 shows TEM image of primaquine phosphate loaded chitosan nanoparticles (A).
In the drawings accompanying the specification, Figure 5 shows SEM image of primaquine phosphate loaded chitosan nanoparticles (B).
In the drawings accompanying the specification, Figure 6 shows AFM image of primaquine phosphate loaded chitosan nanoparticles (C).
In the drawings accompanying the specification, Figure 7 shows FTIR spectra primaquine phosphate (A), chitosan (B), TPP (C), Primaquine phosphate chitosan nanoparticles of F3 (D).
In the drawings accompanying the specification, Figure 8 shows XRD spectrum of primaquine phosphate (A), chitosan (B), TPP (C), primaquine phosphate loaded chitosan nanoparticles (D).
In the drawings accompanying the specification, Figure 9 shows drug release kinetics plots of zero order plot (A), first order plot (B), Higuchi plot (C), Korsmeyer–Peppas plot (D), Hixon Crowell model (E).
Detailed description of the Invention
Accordingly, the present invention provides a formulation of primaquine phosphate - chitosan nanoparticles, comprising of primaquine phosphate; chitosan; and sodium tripolyphosphate; wherein the amount of primaquine phosphate is 10 mg, chitosan is 0.1% and sodium tripolyphosphate is in the range of 0.24 mg/ml to 0.74 mg/ml.
In an embodiment of the invention, the primaquine phosphate loaded chitosan nanoparticles are in the size range of 100 to 900 nm.
In an embodiment of the invention, the polydispersity index (PDI) of primaquine phosphate loaded chitosan nanoparticles is in the range of 0.188 to 0.362.
In an embodiment of the invention, the zeta potential (ZP) of primaquine phosphate loaded chitosan nanoparticles is in the range of 17.2 to 34.2 mV.
The present invention also provides a process for the preparation of primaquine phosphate loaded chitosan nanoparticles, which comprises of dissolving chitosan in an aqueous solution of acetic acid; dissolving primaquine phosphate in chitosan solution; dissolving sodium tripolyphosphate in distilled water; adding sodium tripolyphosphate solution in chitosan drug solution, stirring using magnetic stirrer at 1000 rpm at room temperature for overnight; recovering the nanoparticles by centrifugation of the formulation at 18,000 rpm and collecting supernatant.
Examples
The following examples are given by way of illustration of the present invention and therefore, should not be construed to limit the scope of the present invention.
Materials
All the solvents and chemicals were of analytical grade.
Preparation of primaquine phosphate - chitosan nanoparticles
The primaquine phosphate loaded chitosan nanoparticles were prepared using the modified ionic gelation method. Nanoparticle formation involves an ionic interaction between a positively charged Chitosan solution and a negatively charged sodium tripolyphosphate solution. Various formulation lots of nanoparticles (F1, F2 and F3) were prepared in a constant quantity of Chitosan (1mg/mL) in an aqueous solution of acetic acid (1%). Thereafter, a constant quantity (10 mg) of drug was dissolved directly in 30 ml of chitosan solutions. The sodium tripolyphosphate (TPP) was dissolved in distilled water at a variety of levels (0.24, 0.48, 0.72 mg/ml). After this, 12 ml of each concentration of the aqueous solution of TPP was added in 30 ml of Chitosan solution containing a drug under magnetic stirring (1000 rpm) at room temperature for overnight. The drug loaded Chitosan nanoparticles were collected by centrifugation at 18,000 rpm for 15 min at room temperature and the supernatant was used to assess encapsulation efficiency (EE) (Rawal, T., Parmar, R., Tyagi, R.K. and Butani, S., 2017. Rifampicin loaded chitosan nanoparticle dry powder presents an improved therapeutic approach for alveolar tuberculosis. Colloids and Surfaces B: Biointerfaces, 154, pp.321-330; Tripathy, S., Das, S., Chakraborty, S.P., Sahu, S.K., Pramanik, P. and Roy, S., 2012. Synthesis, characterization of chitosan–tripolyphosphate conjugated chloroquine nanoparticle and its in vivo anti-malarial efficacy against rodent parasite: A dose and duration dependent approach. International journal of pharmaceutics, 434(1-2), pp.292-305).
The composition of the different nanoparticle formulations (F1, F2 and F3) is shown in Table 1.
Table 1. Composition of nanoparticle formulations F1, F2 and F3
S. No. Formulation codes Amount of drug (mg) Chitosan concentration (%) TPP concentration (%)
1. F1 10 0.1 0.24
2. F2 10 0.1 0.48
3. F3 10 0.1 0.72
The prepared nanoparticles were characterized for encapsulation efficiency (EE), particle size, zeta potential (ZP), Transmission Electron Microscopy (TEM), Scanning electron microscopy (SEM), Atomic force microscopy (AFM), Fourier Transform Infrared Spectroscopy (FT-IR), X-ray Diffraction (XRD). The prepared nanoparticles were also characterized for in vitro drug release study using different kinetic models.
The particle size, Zeta potential, PDI and encapsulation efficiency (EE) (%) of nanoparticle formulations F1, F2. F3 are provided in table 2.
Table 2. Particle Size, Zeta Potential, PDI and Encapsulation efficiency (EE) (%) of various nanoparticle formulations
Batches Particle Size (nm) Zeta Potential (mV) PDI Encapsulation efficiency (EE (%)
F1 891.5 17.2 0.362 90
F2 152.3 22.3 0.272 93
F3 233.8 34.2 0.188 97
Encapsulation Efficiency
As shown in Fig. 1, the encapsulation efficiency is 90%, 93% and 97% for nanoparticle formulations, F1, F2 and F3 (Table 2). The highest encapsulation efficiency was observed in batch F3 (97%) at 0.1% chitosan and 0.72% TPP.
Particle Size and Zeta Potential
Primaquine phosphate loaded chitosan nanoparticles formulation F1, F2 and F3 had 891.5 nm, 152.3 nm and 233.8 nm particle size respectively, (Table 2). Table 2 depicts that zeta potential of formulation F1, F2 and F3 are 17.2, 22.3 and 34.2 mV respectively. Nanoparticles F1, F2 and F3 exhibit a polydispersity index of 0.362, 0.272 and 0.188 respectively, (Table 2). Figure 2 shows particle size distribution of nanoparticles, and figure 3 shows zeta potential of nanoparticles.
Morphological Analysis
The shape and surface morphology of the nanoparticles were identified by using TEM, SEM and AFM techniques. The TEM, SEM and AFM image shows the morphological characteristics of optimized nanoparticle formulation (F3). The TEM (Figure 4) and SEM (Figure 5) images revealed spherical particles that were homogeneous and not agglomerated with a relatively narrow size distribution. Atomic force microscopy (Figure 6) provides further information on nanoparticle surface morphology. AFM images of nanoparticles showed that no heterogeneity occurred with the formulation of nanoparticles, and the small average particle size also indicates that the nanoparticles were evenly distributed.
Fourier Transform Infra-red (FTIR) Spectroscopy
Figure 7 shows FTIR spectra of Primaquine phosphate (A), chitosan (B), TPP (C), Primaquine phosphate chitosan nanoparticles F3 (D). FTIR Spectra of Primaquine phosphate showed characteristic peak of aromatic C=C stretching and C-N stretching at 1593.16 cm-1 respectively. Chitosan showed characteristic peak at 3670.57 cm-1 which corresponds to peaks of O–H stretching. The N–H stretching is found to be overlapped in the same region. The stretching frequency of C–O–C and C–N is noted around 1022.78 cm-1,1424.05 cm-1 respectively. The N–H bending and band for amide peak was at 1595 cm-1, 1653 cm-1 respectively. The bending vibration of the NH2 increases from 1653 cm-1 to 1637 cm-1 and 1595 cm-1 to 1570 cm-1 as seen in the spectra of optimized nanoparticle formulation (F3). This can be due to a strong ionic interaction between chitosan amino groups and TPP phosphoric groups. In addition, most of the intense characteristic peaks of drug are not observed at the same position in the optimized formulation of F3 nanoparticles indicating its encapsulation success in the chitosan matrix.
X-ray Diffraction (X-RD)
Figure 8 shows XRD of primaquine phosphate (A), Chitosan (B), TPP (C), Primaquine phosphate loaded chitosan nanoparticles F3 (D). The pure Primaquine phosphate characteristic peaks are seen at 2O of 13.63, 16.64, 18.15, 19.23, 20.33, 22.79, 24.30, 25.11, 27.17, 29.49, 31.53, 33.44. These observed peaks showed that primaquine phosphate exists in a crystalline powder state. The TPP characteristic peak was at 2O of 19.23, 21.97, 24.84, 29.08, 30.03, 31.95, 32.36, 33.44, 34.41, 36.74, 38.24, and 44.51. The chitosan characteristic peaks were at 2O of 20.19, 29.49, 43.02, and 43.97. The acute crystalline peaks of the drug were found to overlap the noise of the coated polymer and disappeared in the diffraction model of the optimized formulation of nanoparticles (F3), suggesting drug was well encapsulated into core of chitosan nanoparticles.
In Vitro Release Studies
The drug release studies were performed for the formulation, produced by modify ionic gelation method by using the dialysis membrane. The optimized formulation, showed high entrapment efficiency, good sustain and controlled release. In vitro drug release took up to 24 hours to complete, as shown in Figures 9.
Figure 9 shows drug release kinetics plots: Zero order plot (A), First order plot (B), Higuchi plot (C), Korsemayer-Pappas plot (D), Hisxon-Crowell plot (E).
To understand the kinetics and the drug release mechanism, the result of the in vitro drug release study of nanoparticles were equipped with various kinetic models like % of drug release vs. time, min. (zero order kinetic model); log cumulative of % drug remaining vs. time, min. (first-order kinetic model); cumulative % drug release vs. square root of time, min. (Higuchi model); log cumulative % drug release vs. log time, min. (Korsmeyer–Peppas model) and cube root of cumulative % drug release vs. time (Hixson-Crowell model).
Table 3. R2 values and rate constants of different models for the in vitro drug release studies.
S. No. Kinetic Model R2 k N
1. Zero Order Model 0.9566 15× 10-1 -
2. First Order Model 0.4045 1.26×10-1 -
3. Higuchi Model 0.9318 1.18 ×10-1 -
4. Hixson-Crowell Model 0.916 0.82×10-3 -
5. Koersemeyer-Peppas Model 0.4662 1.26 × 10-1 0.38
R2 and k-values were calculated for the linear curve produced by regression analysis of the above plots (Table 3). According to the best fit with the maximum regression value (R2), it is concluded that formulated nanoparticles follows the zero order model, the correlation coefficient value of the zero order model was highest, that is 0.9566.