Bioinspired nanocarrier drug delivery system for targeting malaria-infected erythrocytes

Abstract

Currently, we reside in an era of technological innovation and development despite which infectious diseases like malaria remain a threat to humanity. Nanotechnology has entered the realm of drug delivery due to its numerous advantages. Nanocarriers can enhance therapeutic responses, improve drug stability in biological fluids and reduce toxicity by enhancing intracellular penetration and distribution, leading to dose reduction. Additionally, nanoparticles can be surface modified by conjugation with specific ligands to improve active targeting, which will increase selectivity and improve the efficacy of drugs at the site of action. The present research is aimed to develop a targeted antimalarial chemotherapeutic system using human serum albumin (HSA) based nanoparticles as a drug carrier. HSA is a biodegradable protein, nontoxic, nonimmunogenic, and a nutritional requirement for the malarial parasite. Therefore it was considered apt for selective targeting to parasitised red blood cells (pRBCs). Artemether-Human Serum Albumin Nanoparticles (AAN) were prepared by a desolvation method followed by high-pressure homogenisation. To further improve their targetability, AAN were surface modified with pantothenic acid (S1) ligand to yield AAN-S1. Pantothenic acid is a nutritional absolute requirement for parasite growth and development. Blank Albumin nanoparticles (BAN) were prepared as a control. All NPs were lyophilised to improve their physical stability. As NPs <200 nm are reported to be internalised by pRBC the target particle size was set to be <100nm which was achieved with particle size for BAN, AAN and AAN-S1 being 78.57±2.35, 81.54±0.35, 87.57±2.90 nm and polydispersity index 0.49±0.02, 0.46±0.03, 0.04±0.01 respectively as determined by dynamic light scattering. Moreover, the negative zeta potential values -8.49±1.13, -8.37±0.43, -5.66±0.51 mV for the BAN, AAN and AAN-S1 respectively revealed a reasonably stable product. The heat crosslinking in the production method achieved an excellent drug entrapment efficiency of 98.00±0.05 and 97.86±0.02% and drug loading of 13.23±0.03%, and 10.73±0.12% respectively for AAN and AAN-S1 as determined by validated HPLC method. The BAN was found to be stable for six months at both -20, and 4◦C, whereas AAN and AAN-S1 were stable for six months when stored at -20◦C.
The three HSA based NPs displayed a distinct and higher affinity for pRBCs than the uninfected RBCs (uRBCs). They were internalised as early as 5 minutes of incubation. The NPs showed affinity and uptake by all the P. falciparum parasitic blood stages and most of the internalisation happening within 30 minutes. Average uptake by pRBCs was: 80.352.91%, 86.97.82.94%, and 97.552.02% for the BAN, AAN, and AAN-S1 respectively as evaluated by ImageStream flow cytometer. The AAN-S1 showed faster and higher uptake by the ring stage of the parasite as compared to AAN and BAN. Incidentally, growth inhibition studies in P. falciparum K1 strain
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culture for 48 hours showed IC50 values of 11.210.79, 14.771.28, 7.101.28 and 3.860.26 nM for ATM, BAN, AAN, and AAN-S1, correspondingly. Thereby, AAN-S1 exhibited almost three times higher potency as compared to ATM and almost two times higher potency as compared to AAN. An interesting and unexpected finding of this investigation was the antiplasmodial activity exhibited by BAN which can be favourably exploited in future antimalarial chemotherapeutics.
The pRBCs uptake data unravelled that BAN, AAN, and AAN-S1 uptake might be partially energy-dependent. Besides, we showed here a three-fold reduction in AAN-S1 uptake in the presence of furosemide, a New Permeability Pathways (NPP) inhibitor, suggesting NPPs as one of the major pathways for the internalisation of AAN-S1. Remarkably, BAN, AAN, and AAN-S1 showed a range of 0.61±0.01 to 1.31±0.19% uptake in uRBCs indicating these NPs might have a higher affinity for pRBCs than uRBCs; which might further confirm their safety demonstrated by our preliminary cytotoxicity study. The in vitro cytotoxicity on HepG2 cells revealed the NPs to be safe as determined by IC80 values of 25 ±2.09, 300 ±1.11, 75 ±1.36, and 100 ±1.64 μM for ATM, BAN, AAN, and AAN-S1 respectively. IC50 values could not be determined even after incubation at 1000 μM concentration for 72 h.
This work might provide a starting point for a future nano-combination for malarial therapy. At this point, it is worth introducing the aspect of albumin-based nanocarriers as a potential nanocarrier targeted delivery tool for malaria therapy, to enable a better understanding of upcoming works. In this regard, the unusual antiplasmodial activity of BAN that was unveiled in this study might make it not only a potential drug carrier for an antimalarial drug but, a potential candidate for prevention, treatment, and diagnosis of malaria.