INTRODUCTION:

Drug delivery systems based on nanoparticles (DDS) have been effectively used in trials and clinical settings in recent decades to increase the effectiveness of numerous medications and therapeutic molecules^1,2Because these DDSs have an enhanced permeability and retention (EPR) effect, they can passively target tumours.^3,4 medicines based on nanoparticles have several advantages over free medicines, including reduced toxicity, excellent stability, exceptional biocompatibility, increased drug release efficiency, longer blood retention times, and extended shelf lives for biomedical purposes.⁵

Nevertheless, ideal nanoparticles should also include characteristics like increased blood circulation and tumour tissue, particularly for cancer treatments. targeting as well as binding to cancer cells.

Many types of cell membranes, including as those from bacteria, lymphocytes, platelets, leukocytes, cancer cells, and erythrocytes (red blood cells, or RBCs), have been inspired by nature to achieve long-term circulation or tumour targeting. enabling blood flow RBCs were first investigated for up to 120 days and have been utilized as an perfect vehicle for delivering a range of bioactive substances, including medications, macromolecules, proteins, and enzymes⁶ Furthermore, mature RBCs are ideal for extraction and purification since they are devoid of a nucleus and other organelles.⁷Human blood is the most abundant tissue in the body, with an average of 5 billion red blood cells per millilitre. This makes blood a rich source of coating materials for drug carriers. the process of functionalization. Biconcave discs consisting of RBCs average 1. μm thick in the centre, 2.5μm on the margin, and 7.8μm in diameter 85–91 fl (μm3) volume⁸ RBCs eventually turn spherical as the surrounding medium's osmotic pressure decreases. The viability of injecting medications or other substances into RBCs depends on this swelling characteristic. To summarize, RBCs are advantageous when used as carriers for loading nanoparticles because^8-10 a) long-term circulation and immune system evasion; b) notable intrinsic biocompatibility and biodegradability; and c) avoiding certain inherent toxicity associated with nanopreparation; d) a 120-day lifetime; e) Because of the vast amounts, easily reaching high load capacity of cell membranes; and f) strengthening the stability of nanoparticles and reducing aggregation and extending the in vitro storage period. Notably, anticancer research has made significant progress with the widespread adoption of erythrocyte membrane-coated nano-formulations.^11-13Certain characteristics of red blood cells, like their structure and surface proteins, have also been used as design cues to create the next wave of drug delivery systems.^14-16 We restrict our attention to several facets of the field of erythrocyte membrane-coated nano-cores in this review, with a special emphasis on coating. procedures, techniques for getting ready, techniques for checking, and the most recent anti-tumor software. Modern developments offer assurance. in the foreseeable future towards their clinical use. Nevertheless, due to This platform is made of biological components that have undergone rigorous cleaning. and accurate blood type matching are necessary to optimise harmony and minimise the chance of immunogenicity.

History:

The Dutch scientist Lee Van Hock first described red blood cells (RBCs) in human blood samples in the 17th century. Howson discovered that RBCs were flat discs rather than spherical shapes a century later. In 1953, Gardos made an attempt to inject ATP into the "erythrocyte ghosts." with this endeavour providing the groundwork for a later covering of the erythrocyte membrane with different active components, creating a entire new field of medication administration techniques. 1959 saw Marsden and Ostling¹⁷ Ihler et al. first described the trapping of dextrans in erythrocytes and then used RBC loading with therapeutic drugs for delivery.⁶ The phrase "carrier red blood cells" was subsequently coined in 1979.¹⁸

After a ground-breaking study in the 1970s employing carrier erythrocytes to treat Gaucher's disease with β-glucosidase and β-galactosidase⁶ Numerous enzyme replacement therapy approaches have been created, including the loading of L-asparaginase into erythrocytes, daunorubicin, and gentamicin to treat leukaemia, bacterial infections, and asparagine-dependent leukaemia¹⁹ Lejeune et al., 1994²⁰ described the process of creating "nanoerythrosomes," or liposomes produced from RBC membranes, by manually pressing RBC ghosts through membranes having specific pore diameters. But only conjugating active compounds onto erythrocyte surfaces has been discovered to cause both a quick removal from the bloodstream and a propensity to induce vesicle aggregation, suggesting that these very unstable erythrocyte ghosts lack structural integrity²¹ In 2013, the Zhang team²²provided a biomimetic poison nanosponge that acted as a toxin decoy in vivo and defined nanosponges. The nanosponges, which are made up of polymer nanoparticle cores and erythrocyte membranes surrounding them, have the ability to absorb toxins that damage membranes and then move them away from their intended target cells. Regarded as one of this field's most fascinating discoveries, This work sparked a surge in research on bionic nanomedicine.

Fig 1- The historical process of RBCs as drug carriers

Preparation Method of RBCM- NPs:

Several approaches, such as those based on chemical and physical properties, have been documented for encasing medications or other bioactive substances in erythrocytes, such as hypotonic hemolysis.^23,24 a hypotonic dilution^25,26 dialysis that is hypotonic²⁷ hypotonic pre-inflation^28,29 and the osmotic pulse³⁰or the membrane's chemical disturbance^31,32 in addition to electrical failure³³ Different molecules can also be encapsulated by erythrocytes through endocytosis, lipid fusion, and intrinsic absorption.⁸ The encapsulated chemicals may need a significant level of water solubility in addition to erythrocyte inactivity- that is, absence of interacts with erythrocyte membranes both chemically and physically to Prevent the laden RBCs from leaking, as this could lead to toxicological issues. Consequently, as the foundation, medications are frequently made into a nano preparation with reduced toxicity and increased stability, followed by disguising the nano-preparations with RBCMs in order to prevent immune systems' recognition.

1. Preparation of RBCM-derived vesicles (RVs):

The two main components of the optimized and widely used synthesis of RBCM-NPs are vesicle-particle fusion and membrane-derived vesicles from RBCs.^28,32,34 Sequential extrusion and hypotonic therapy are used to create RVs. After being extracted from the organism (such as a mouse), fresh whole blood is centrifuged at 41C to sustain protein activity, following which the buffy coat and serum are taken out to gather erythrocytes. The ensuing RBCs are consistently cleaned with phosphate-buffered saline (PBS) and then gathered again by centrifugation to get rid of extraneous cells and leftover plasma. After that, RBC ghosts are obtained using hypotonic therapy, in which the Washing RBCs are combined with an excess of 0.25 PBS very gently.[^34] and held to release the components of the intracellular RBC. The RBC ghosts that make up the pink precipitate after high-speed centrifugation are sonicated in a bath so nicator and then passed through polycarbonate porous membranes with varying pore sizes using an Avanti mini-extruder to produce the desired size of RVs. (Figure 2). In order to maintain the membrane's bioactivity, protease inhibitors often included with the samples, which are then kept at 41C.^35-37

Fig 2: Diagram showing how red blood cell membrane-derived vesicles (RVs) are prepared. To obtain clean RBCs, fresh whole blood was centrifuged and extensively washed. Subsequently, RVs were obtained by additional hypotonic and extrusion treatment.

2. Coating mechanism of RBCM-NPs:

RBCs can flow through relatively small capillary networks because of their highly flexible shape, which is dependent on the viscoelasticity of the cell membrane, the viscosity of the cell content, and the ratio of the cell surface to volume. and "sieving organs" like the liver and spleen³⁸. The glycocalyx, a thick layer of polysaccharides on the surface of red blood cells, is crucial for immune escape traits and cell stability.^39,40 In order to achieve spatial stability, these complex polysaccharides on the cell surface are comparable to a hydrophilic coating.^41,42Higher surface energy polymeric nanoparticles are more likely to interact with the polysaccharide's stabilized membranes to reduce total energy, however the RBCM-NP surface that has stabilized can exclude additional interactions between membranes⁴³ Even in the presence of excess RBCMs, this stabilization process guarantees the occurrence of monolayer film coating.

Additionally, the silyl residues in the negatively charged the polysaccharide terminal gives the cell a charged asymmetry. membranes, which are essential for interfacial interactions between nanoparticles and RBCMs. Examination of the positive and RBCMcoated negatively charged nanoparticles by Luk et al.⁴³ showed that although positively charged nanoparticles only created polydisperse aggregates, negatively charged nanoparticles were able to build nuclei-shells with unique particles. (Figure 3). These outcomes might be explained by the dense negatively the side of the outer membrane's charged sialic acid moiety, with the strong attraction from positively charged nanoparticles probably causes the lipid bilayer to collapse and prevents the local arrangement from happening. essential for the coverage of lipids. Conversely, the negatively charged Strong electrostatic repulsion between the sialic acid moiety and the nanoparticles led to the nanoparticles fusing with the intracellular membrane side to form a structure with a rightside-out membrane orientation.⁴⁴ help keep the glycocalyx on the cell surface.

Fig 3: Diagram illustrating the electrostatic interactions of RVs that are negatively and asymmetrically charged and have polymeric cores that are negatively and positively charged, respectively. Strong electrostatic repulsion between the negatively charged nanoparticles and the negatively charged RVs causes the nanoparticles to fuse with the intracellular membrane side, whereas the positively charged nanoparticles and the negatively charged RVs that are negatively charged have a significant aptitude for breaking down the lipid bilayer. Reproduced with permission ⁴³

3. Methods of Vesicle-Particle Fusion:

Using "bottom-up" techniques, or functionalizing nanoparticles with RBC surface chemistry, was the focus of early attempts to bridge nanoparticles and RBCMs. But the RBC-mimicking delivery systems created via chemistry-based bioconjugation frequently cause denaturation of proteins. To further anchor RBCMs, the nanoparticles could scarcely be enough replicated into a complicated protein. 2011 saw Zhang and colleagues.^[45] revealed a "top-down" technique for creating nanoparticles with RBCM camouflage. They were able to successfully coat the sub-100-nm PLGA nanoparticles with the RBCMs by extruding nanoparticles with nanoscale RVs that were prepared beforehand. There is hope for the large-scale production of RBCM-NPs using this "top-down" approach. A summary of the main techniques for RV-nanoparticle fusion can be found here.

A) Co-Extrusion Method:

Typically, prepared nanoparticles and obtained RVs are fused via mechanical extrusion. The aforementioned interfacial interactions serve as the coating process's guiding principle. The mixture is repeatedly extruded through various sized porous membranes, depending on the size of the prepared nanoparticles, and then bath sonicated for a few minutes.⁴³ (Figure 4a) The vesicle-particle fusion is produced by the mechanical force pushing the nanoparticles through the lipid bilayer. To reduce membrane protein loss and degradation, the RBCM phospholipid bilayer structure in particular needs to be as complete as possible during the preparation process.⁴⁵ Following several extrusions, the extra vesicles are eliminated by centrifugation, and the final product is represented by the precipitate that is collected and redispersed for later use.³⁴ Based on the volume of the RBC membrane and the total membrane volume needed to fully coat 1 mg of nanoparticles, the ratio of RBCMs to nanoparticles is calculated. Brian et al.^43,45 employed various PLGA nanoparticle sizes, ranging in diameter from 65 to 340nm, and coated them with erythrocyte membranes, producing varying quantities of erythrocyte membranes as needed. The fewer red blood cell membranes required for a given weight of nanoparticles, the smaller the particle size. Excessive volumes of blood are typically required to ensure that all nanoparticles are coated with RBCMs, making up for cell membrane loss during preparation and the fusion process with nanoparticles.⁴⁴

Fig 4-Co-extrusion Method

Fig 5-Microfluidic Electroporation Method

B) Microfluidic Electroporation Method:

The biomedical field has been using biomimetic membrane-coated nanoparticles for a long time. Microfluidic electroporation has also been shown to be an effective method for promoting RBCM-NP synthesis. Rao and associates⁴⁶ Fe3O4 magnetic nanoparticles (MNs) in combination with RVs in a microfluidic device. Five sections make up the microfluidic chip for electroporation: an electroporation zone, an S-shaped mixing channel, a Y-shaped merging channel, two inlets for the nanoparticles and RVs, respectively, and an outlet. Multiple transient pores can be created and the dielectric layer on the cell membranes broken down by electrical pulses when the mixture of MNs and RVs passes through the electroporation zone.⁴⁷ allowing a channel for Fe3O4 MNs to enter. The pulse voltage, duration, and flow rate should all be optimized during this process. Following integration, the RBCM-MN are extracted from the chips and introduced into test animals in order to conduct a number of in vivo functional assessments (Fig. 4B)

Unlike the co-extrusion method, the microfluidic electroporation strategy perfectly combines biology and physics. It achieves a better therapeutic effect by reducing cell surface protein loss and maintaining some membrane integrity by eliminating the need for a very large force to repeatedly squeeze the nanoparticles through porous membranes. Furthermore, compared to those made using traditional extrusion techniques, RBCM-MNs made via microfluidic electroporation demonstrate superior colloidal stability, magnetic resonance imaging (MRI), and photothermal therapy (PTT) performance in vivo. Consequently, allowing a channel for Fe3O4 MNs to enter. The pulse voltage, duration, and flow rate should all be optimized during this procedure. Following integration, the RBCM-MN are extracted from the chips and administered intraperitoneally to the test animals. Therefore, there seem to be promising prospects for using the microfluidic electroporation approach to coat nanoparticles in a bioinspired cell membrane.

4. In vitro Verification of RBCM-NPs:

Due to the critical roles that the RBCMs' surface proteins and chemical structures play in their immunological escape and circulation; an in vitro study of these cellular vectors is required. The following are the primary characterization parameters.

A) UV–vis Absorption Spectra:

UV–vis absorption spectroscopy is another tool for characterizing encapsulation conditions. The encapsulated nanostructures maintain the original absorption peak in comparison to the original nanoparticle absorption pattern, and they also get an additional absorption peak that is equivalent to the distinctive RV absorption peak.^34,48 This outcome shows that the RVs were successfully shifted onto the nanoparticle surface without changing the original nanoparticles' characteristics. When taken as a whole, these in vitro analyses confirm that RBC integration with nanoparticles and vesicle separation are optimal.

B) Fluorescence Colocalization:

Hu et al. developed a procedure wherein hydrophobic red DiD dyes⁴⁵and lipophilic green rhodamine-DMPE dyes were inserted into the polymeric cores and RVs, respectively, before their fusion, in order to further confirm the integrity of the core-shell particle structure. Via the use of a fluorescent microscope, the dual-fluorophore-labeled nanoparticles were found to overlap at the same location after being treated with HeLa cells for 6 hours. As the nanoparticles were internalized by the cells, fluorescence colocalization revealed that they possessed a full core-shell structure, proving the effectiveness of the RBCM coating.

CONCLUSION:

An innovative biomimetic approach to targeted medication administration, the use of erythrocyte membrane-coated nanoparticles shows great promise for improving therapeutic results. This novel strategy successfully eludes the immune system, promotes sustained circulation, and permits targeted therapy of illnesses, including cancer, by utilising the biological interactions of cell membranes. Even if there are still problems with scalability, quality control, regulations, and batch-to-batch fluctuation, these can be resolved with continued study and development. Its broad potential is highlighted by the technology's adaptability, which includes different cell membranes for particular disease applications. The combination of natural and synthetic biomaterials has the potential to transform drug delivery and treatment approaches as this field develops further, giving patients fresh hope for better results.

REFERENCES:

1. Kang L, Gao Z, Huang W, Jin M, Wang Q. Nanocarrier-mediated co-delivery of chemotherapeutic drugs and gene agents for cancer treatment. Acta Pharm Sin B. 2015; 5: 169–75.

2. Weng Y, Liu J, Jin S, Guo W, Liang X, Hu Z. Nanotechnology-based strategies for treatment of ocular disease. Acta Pharm Sin B. 2017; 7:281–91.

3. Fang J, Qin H, Nakamura H, Tsukigawa K, Shin T, Maeda H. Carbon monoxide, generated by heme oxygenase-1, mediates the enhanced permeability and retention effect in solid tumors. Cancer Sci. 2012; 103:535–41.

4. Iwamoto T. Clinical application of drug delivery systems in cancer chemotherapy: review of the efficacy and side effects of approved drugs. Biol Pharm Bull. 2013; 36:715–8.

5. Chen MC, Lin ZW, Ling MH. Near-infrared light-activatable microneedle system for treating superficial tumors by combination of chemotherapy and photothermal therapy. ACS Nano. 2016; 10:93–101.

6. Ihler GM, Glew RH, Schnure FW. Enzyme loading of erythrocytes. Proc Natl Acad Sci USA. 1973; 70:2663–6.

7. Pierigè F, Serafini S, Rossi L, Magnani M. Cell-based drug delivery. Adv Drug Deliv Rev. 2008; 60: 286–95.

8. Hamidi M, Tajerzadeh H. Carrier erythrocytes: an overview. Drug Deliv. 2003; 10: 9–20.

9. Patel PD, Dand N, Hirlekar RS, Kadam VJ. Drug loaded erythrocytes: as novel drug delivery system. Curr Pharm Des. 2008; 14: 63–70.

10. Dehaini D, Wei X, Fang RH, Masson S, Angsantikul P, Luk BT, et al. Erythrocyte-platelet hybrid membrane coating for enhanced nanoparticle functionalization. Adv Mater. 2017; 29: 1606209.

11. Yoo JW, Irvine DJ, Discher DE, Mitragotri S. Bio-inspired, bioengineered and biomimetic drug delivery carriers. Nat Rev Drug Discov. 2011; 10: 521–35.

12. Wang C, Sun X, Cheng L, Yin S, Yang G, Li Y, et al. Multifunctional theranostic red blood cells for magnetic-field-enhanced in vivo combination therapy of cancer. Adv Mater. 2014; 26: 4794–802.

13. Sun X, Wang C, Gao M, Hu A, Liu Z. Remotely controlled red blood cell carriers for cancer targeting and near-infrared lighttriggered drug release in combined photothermal-chemotherapy. Adv Funct Mater. 2016; 12: 548

14. Doshi N, Zahr AS, Bhaskar S, Lahann J, Mitragotri S. Red blood cell-mimicking synthetic biomaterial particles. Proc Natl Acad Sci USA. 2009; 106: 21495–9.

15. Tsai RK, Rodriguez PL, Discher DE. Self inhibition of phagocytosis: the affinity of 'marker of self' CD47 for SIRP alpha dictates potency of inhibition but only at low expression levels. Blood Cells Mol Dis. 2010; 45: 67–74.

16. Merkel TJ, Jones SW, Herlihy KP, Kersey FR, Shields AR, Napier M, et al. Using mechanobiological mimicry of red blood cells to extend circulation times of hydrogel microparticles. Proc Natl Acad Sci USA. 2011; 108:586–91.

17. Marsden NV, Ostling SG. Accumulation of dextran in human red cells after haemolysis. Nature. 1959; 184(Suppl 10): 723–4.

18. Jain S, Jain NK. Engineered erythrocytes as a drug delivery system. Indian J Pharm Sci. 1997; 59: 275–81.

19. Hu CM, Fang RH, Zhang L. Erythrocyte-inspired delivery systems. Adv Healthc Mater. 2012; 1: 537–47.

20. Lejeune A, Moorjani M, Gicquaud C, Lacroix J, Poyet P, Gaudreault R. Nanoerythrosome, a new derivative of erythrocyte ghost: preparation and antineoplastic potential as drug carrier for daunorubicin. Anticancer Res. 1994; 14: 915–9.

21. Desilets J, Lejeune A, Mercer J, Gicquaud C. Nanoerythrosomes, a new derivative of erythrocyte ghost: iv. Fate of reinjected nanoerythrosomes. Anticancer Res 2001; 21:1741–7.

22. Hu CM, Fang RH, Copp J, Luk BT, Zhang L. A biomimetic nanosponge that absorbs pore-forming toxins. Nat Nanotechnol. 2013; 8: 336–40.

23. Ihler GM, Tsang HC. Hypotonic hemolysis methods for entrapment of agents in resealed erythrocytes. Methods Enzymol. 1987; 149:221–9.

24. Sato Y, Yamakose H, Suzuki Y. Mechanism of hypotonic hemolysis of human erythrocytes. Biol Pharm Bull. 1993; 16:506–12.

25. Seth M, Ramachandran A, Leal LG. Dilution technique to determine the hydrodynamic volume fraction of a vesicle suspension. Langmuir. 2010; 26: 15169–76.

26. Bird J, Best R, Lewis DA. The encapsulation of insulin in erythrocytes. J Pharm Pharmacol. 1983; 35:246–7.

27. Gutierrez MC, Zarzuelo CA, Sayalero MM, Lanao JM. Factors associated with the performance of carrier erythrocytes obtained by hypotonic dialysis. Blood Cells Mol Dis. 2004; 33: 132–40

28. Tajerzadeh H, Hamidi M. Evaluation of hypotonic preswelling method for encapsulation of enalaprilat in intact human erythrocytes. Drug Dev Ind Pharm. 2000; 26: 1247–57.

29. Hamidi M, Zarrin AH, Foroozesh M, Zarei N, Mohammadi-Samani S. Preparation and in vitro evaluation of carrier erythrocytes for REStargeted delivery of interferon-α2b. Int J Pharm. 2007; 341: 125–33.

30. Franco RS, Barker R, Novick S, Weiner M, Martelo OJ. Effect of inositol hexaphosphate on the transient behavior of red cells following a DMSO-induced osmotic pulse. J Cell Physiol. 1986; 129: 221–9.

31. Watts TJ, Handy RD. The haemolytic effect of verapamil on erythrocytes exposed to varying osmolarity. Toxicol Vitr. 2007; 21: 835–9.

32. Lynch AL, Chen R, Slater NK. pH-responsive polymers for trehalose loading and desiccation protection of human red blood cells. Biomaterials. 2011; 32: 4443–9.

33. Choi SO, Kim YC, Park JH, Hutcheson J, Gill HS, Yoon YK, et al. An electrically active microneedle array for electroporation. Biomed Microdevices. 2010; 12: 263–73.

34. Ren X, Zheng R, Fang X, Wang X, Zhang X, Yang W, et al. red blood cell membrane camouflaged magnetic nanoclusters for imaging-guided photothermal therapy. Biomaterials. 2016; 92: 13–24.

35. Zhai Y, Su J, Ran W, Zhang P, Yin Q, Zhang Z, et al. Preparation and application of cell membrane-camouflaged nanoparticles for cancer therapy. Theranostics. 2017; 7:2575–92.

36. Hu CM, Fang RH, Wang KC, Luk BT, Thamphiwatana S, Dehaini D, et al. Nanoparticle biointerfacing by platelet membrane cloaking. Nature. 2015; 526: 118–21.

37. Su J, Sun H, Meng Q, Yin Q, Tang S, Zhang P, et al. long circulation red-blood-cell mimetic nanoparticles with peptide-enhanced tumor penetration for simultaneously inhibiting growth and lung metastasis of breast cancer. Adv Funct Mater. 2016; 26: 1243– 52.

38. Fang RH, Hu CM, Zhang L. Nanoparticles disguised as red blood cells to evade theimmune system. Expert Opin Biol Ther. 2012; 12: 385–9.

39. Schauer R. Sialic acids as regulators of molecular and cellular interactions. Curr Opin Struct Biol. 2009; 19: 507–14.

40. Kelm S, Schauer R. Sialic acids in molecular and cellular interactions. Int Rev Cytol. 1997; 175: 137–240.

41. Raveendran P, Fu J, Wallen SL. Completely “green” synthesis and stabilization of metal nanoparticles. J Am Chem Soc. 2003; 125: 13940–1.

42. Lemarchand C, Gref R, Couvreur P. Polysaccharide-decorated nanoparticles. Eur J Pharm Biopharm. 2004; 58: 327–41.

43. Luk BT, Hu CM, Fang RH, Dehaini D, Carpenter C, Gao W, et al. Interfacial interactions between natural RBC membranes and synthetic polymeric nanoparticles. Nanoscale. 2014; 6: 2730–7.

44. Hu CM, Fang RH, Luk BT, Chen KN, Carpenter C, Gao W, et al. ‘Marker-of-self’ functionalization of nanoscale particles through a topdown cellular membrane coating approach. Nanoscale. 2013; 5: 2664–8.

45. Hu CM, Zhang L, Aryal S, Cheung C, Fang RH, Zhang L. Erythrocyte membrane camouflaged polymeric nanoparticles as a biomimetic delivery platform. Proc Natl Acad Sci USA. 2011; 108: 10980–5.

46. Rao L, Cai B, Bu LL, Liao QQ, Guo SS, Zhao XZ, et al. Microfluidic electroporation facilitated synthesis of erythrocyte membrane-coated magnetic nanoparticles for enhanced imaging guided cancer therapy. ACS Nano. 2017; 11: 3496–505.

47. Miyazaki J, Aihara H. Gene transfer into muscle by electroporation in vivo. Methods Mol Med. 2002; 69: 49–62.

48. Wang X, Li H, Liu X, Tian Y, Guo H, Jiang T, et al. Enhanced photothermal therapy of biomimetic polypyrrole nanoparticles through improving blood flow perfusion. Biomaterials. 2017; 143: 130–41.

Received on 13.11.2025 Revised on 10.12.2025

Accepted on 03.01.2026 Published on 13.04.2026

Available online from April 15, 2026

Asian J. Pharm. Tech. 2026; 16(2):201-206.

DOI: 10.52711/2231-5713.2026.00029

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. Creative Commons License.