Chemistry and Art of Developing Lipid Nanoparticles for Biologics Delivery: Focus on Development and Scale-Up.

Lipid nanoparticles (LNPs) have gained prominence as primary carriers for delivering a diverse array of therapeutic agents. Biological products have achieved a solid presence in clinical settings, and the anticipation of creating novel variants is increasing. These products predominantly encompass therapeutic proteins, nucleic acids and messenger RNA. The advancement of efficient LNP-based delivery systems for biologics that can overcome their limitations remains a highly favorable formulation strategy. Moreover, given their small size, biocompatibility, and biodegradation, LNPs can proficiently transport therapeutic moiety into the cells without significant toxicity and adverse reactions. This is especially crucial for the existing and upcoming biopharmaceuticals since large molecules as a group present several challenges that can be overcome by LNPs. This review describes the LNP technology for the delivery of biologics and summarizes the developments in the chemistry, manufacturing, and characterization of lipids used in the development of LNPs for biologics. Finally, we present a perspective on the potential opportunities and the current challenges pertaining to LNP technology.

CRISPR/Cas9 Genome Editing for Tissue-Specific In Vivo Targeting: Nanomaterials and Translational Perspective.

Clustered randomly interspaced short palindromic repeats (CRISPRs) and its associated endonuclease protein, i.e., Cas9, have been discovered as an immune system in bacteria and archaea; nevertheless, they are now being adopted as mainstream biotechnological/molecular scissors that can modulate ample genetic and nongenetic diseases via insertion/deletion, epigenome editing, messenger RNA editing, CRISPR interference, etc. Many Food and Drug Administration-approved and ongoing clinical trials on CRISPR adopt ex vivo strategies, wherein the gene editing is performed ex vivo, followed by reimplantation to the patients. However, the in vivo delivery of the CRISPR components is still under preclinical surveillance. This review has summarized the nonviral nanodelivery strategies for gene editing using CRISPR/Cas9 and its recent advancements, strategic points of view, challenges, and future aspects for tissue-specific in vivo delivery of CRISPR/Cas9 components using nanomaterials.

Nanocrystals for controlled delivery: state of the art and approved drug products.

INTRODUCTION: Controlled/extended-release formulations offer numerous benefits over conventional especially reduced side effects, improved therapeutic outcomes, and high patient compliance. Controlled release nanocrystal is extremely versatile technology with several advantages such as very high drug loading, ease of manufacturing, avoidance of dose dumping, reproducible drug release. Usually, nanonization of drug is performed to improve dissolution rate, intrinsic solubility, and thereby bioavailability. Most of the times, this is done for immediate release dosage forms where objective is quick onset of action. However, nanocrystals can also provide a sustained, reproducible plasma concentration profile for weeks to months based on tissue microenvironment, surface coating and administration route. AREAS COVERED: This review briefly describes the methods for producing nanocrystals, summarizes preclinical research and commercial products demonstrating tremendous potential of controlled release nanocrystals. EXPERT OPINION: Lipophilic drugs are attractive candidates for the development of nanocrystal based controlled release formulations. However, constraint should be practiced while generalizing the technology for the controlled release purpose. Not all drugs fit in the requirement from the perspectives of physicochemical properties or pharmacokinetics. Additionally, technologies should be explored which can convert the nanocrystal into its final dosage form for administration yet preserves the benefits of small particle size and controlled release.

Stromal disruption facilitating invasion of a 'nano-arsenal' into the solid tumor.

Owing to the indispensable role of nanotechnology in cancer therapy, it is imperative to comprehend every aspect limiting its therapeutic potential. Several preclinical reports have demonstrated the enhanced permeability and retention (EPR)-mediated preferential tumor uptake of nanoparticles. However, the therapeutic outcome of nanotherapeutics is severely compromised by heterogeneous drug distribution and insufficient penetration of nanomedicine in a solid tumor owing to the dense tumor extracellular matrix (ECM). Herein, we elaborate on various preclinically investigated tumor stromal disrupting strategies, which we call 'cannons', to compromise the impenetrable 'fortress-like' solid tumor microenvironment. We have described and summarized major approaches to enhance the penetration of a 'nano-arsenal' in solid tumors. ECM remodeling strategies could be very beneficial in enhancing the therapeutic efficacy of monoclonal antibodies and translational nanomedicine.

COVID-19 associated complications and potential therapeutic targets.

The global pandemic COVID-19, caused by novel coronavirus SARS-CoV-2, has emerged as severe public health issue crippling world health care systems. Substantial knowledge has been generated about the pathophysiology of the disease and possible treatment modalities in a relatively short span of time. As of August 19, 2020, there is no approved drug for the treatment of COVID-19. More than 600 clinical trials for potential therapeutics are underway and the results are expected soon. Based on early experience, different treatment such as anti-viral drugs (remdesivir, favipiravir, lopinavir/ritonavir), corticosteroids (methylprednisolone, dexamethasone) or convalescent plasma therapy are recommended in addition to supportive care and symptomatic therapy. There are several treatments currently being investigated to address the pathological conditions associated with COVID-19. This review provides currently available information and insight into pathophysiology of the disease, potential targets, and relevant clinical trials for COVID-19.

Exploring molecular dynamics simulation to predict binding with ocular mucin: An in silico approach for screening mucoadhesive materials for ocular retentive delivery systems.

In recent times, molecular dynamic (MD) simulations have been applied in the area of drug delivery, as an in silico tool to predict the behaviour of nanoparticles with respect to their interaction with larger biological entities like bilayer membranes, DNA and dermal surface. However, the predictions must be systematically evaluated by extensive studies with actual biological entities in order to deem the in silico models accurate. Thus, in the present study, MD simulation was used to screen ligands with respect to ocular mucoadhesion. Mucin-4, a cell surface-associated mucin was selected as the substrate for the in silico study due to its abundance across the ocular surface. The ligands were then incorporated into a delivery system like nanostructured lipid carriers (NLC) and assessed for mucoadhesion by relevant in vitro and in vivo techniques. The in silico study suggested chitosan oligosaccharide (COS) to have an extensive mucoadhesive potential towards ocular mucin followed by stearylamine (STA) and cetrimonium bromide (CTAB) which showed intermediate and low mucoadhesion respectively. The corresponding in vitro assessment by spectrophotometry and nanoparticle tracking analysis showed a similar outcome wherein COS was found to be extensively mucoadhesive, followed by both STA and CTAB, which showed mucoadhesion to a nearly equal extent. The findings of in vivo confocal imaging following topical administration to rats showed that while COS and STA adhered extensively to the ocular surface, CTAB showed negligible adhesion. MD simulation was thus found to accurately predict interactions critical to mucoadhesion and the same could be fairly correlated well by relevant mucoadhesion studies both in vitro and in vivo.

Cationic cholesterol derivative efficiently delivers the genes: in silico and in vitro studies.

The aims of the research work were to synthesize ethyl(cholesteryl carbamoyl)-L-arginate (ECCA), an arginine-conjugated cholesterol derivative, and to evaluate its application as a gene delivery vector. The interactions of ECCA with DNA duplex were studied using molecular dynamics (MD) simulations. It was found that the guanidine group of ECCA could interact with the phosphate group of DNA through ionic interactions as well as hydrogen bonds. The structure of DNA was stable throughout the simulation time. Liposomes were formulated using ECCA and soya phosphatidylcholine (SPC) by a thin-film hydration method. They had the particle size of ~ 150 nm and the zeta potential of + 51 mV. To ensure the efficient binding of DNA to the liposomes, the ratio of DNA to ECCA was optimized using gel retardation assay. Further, serum stability, haemolysis and cytotoxicity studies were carried out to determine the stability and safety of the lipoplexes. Circular dichroism spectroscopy was used to determine the interaction of DNA and cationic liposomes. Cellular uptake pathway was determined by studying the uptake of coumarin-loaded lipoplexes at 4 °C and in the presence of uptake inhibitors, i.e. genistein, chlorpromazine and methyl-ß-cyclodextrin. Transfection studies were carried out to evaluate the transfection efficacy of the ECCA-loaded lipoplexes. The binding of DNA and lipoplexes was found to be stable in the presence of serum, and no degradation of DNA was observed. The lipoplexes showed low haemolysis and cytotoxicity. The uptake of coumarin-loaded liposomes was decreased up to ~ 20% in the presence of clathrin- and caveola-mediated uptake inhibitors, indicating a role of both the pathways in the uptake of the inhibitors. Satisfactory transfection efficiency was obtained compared to Lipofectamine®. Thus, cationic cholesterol derivative is a useful tool for gene delivery vector.

Liposils: An effective strategy for stabilizing Paclitaxel loaded liposomes by surface coating with silica.

The present work aims at improving stability of paclitaxel (PTX) loaded liposomes by its coating with silica on the surface by a modified sol-gel method. Effect of various components of liposomes such as phosphatidylcholine to cholesterol ratio (PC:CH), PTX and stearylamine on entrapment efficiency (% EE) and particle size were systematically investigated and optimized using central composite design on Design-Expert®. The optimized liposomes were utilized as a template for silica coating to prepare surface coated PTX liposils. Physical stability of liposomes and liposils was evaluated with Triton X-100 and the results indicated that liposils were much more stable as compared to liposomes and the same has been reiterated in stability study performed over 6 months. In vitro cytotoxicity study on B16F10 tumor cells showed cytotoxicity of PTX liposils was not significantly different than PTX liposomes, whereas both were less cytotoxic as compared to the commercial Taxol®. In vivo pharmacokinetics on rats, exhibited increased T1/2 of liposils when compared to liposomes and Taxol®, thus releasing the drug over a longer duration. The enhanced physicochemical stability as well as controlled release of PTX in liposils developed in this study could be an effective alternative to Taxol® and PTX liposomes.

Rational Design of Cholesterol Derivative for Improved Stability of Paclitaxel Cationic Liposomes.

PURPOSE: This work explores synthesis of novel cholesterol derivative for the preparation of cationic liposomes and its interaction with Paclitaxel (PTX) within liposome membrane using molecular dynamic (MD) simulation and in-vitro studies. METHODS: Cholesteryl Arginine Ethylester (CAE) was synthesized and characterized. Cationic liposomes were prepared using Soy PC (SPC) at a molar ratio of 77.5:15:7.5 of SPC/CAE/PTX. Conventional liposomes were composed of SPC/cholesterol/PTX (92:5:3 M ratio). The interaction between paclitaxel, ligand and the membrane was studied using 10 ns MD simulation. The interactions were studied using Differential Scanning Calorimetry (DSC) and Small Angle Neutron Scattering analysis. The efficacy of liposomes was evaluated by MTT assay and endothelial cell migration assay on different cell lines. The safety of the ligand was determined using the Comet Assay. RESULTS: The cationic liposomes improved loading efficiency and stability compared to conventional liposomes. The increased PTX loading could be attributed to the hydrogen bond between CAE and PTX and deeper penetration of PTX in the bilayer. The DSC study suggested that inclusion of CAE in the DPPC bilayer eliminates Tg. SANS data showed that CAE has more pronounced membrane thickening effect as compared to cholesterol. The cationic liposomes showed slightly improved cytotoxicity in three different cell lines and improved endothelial cell migration inhibition compared to conventional liposomes. Furthermore, the COMET assay showed that CAE alone does not show any genotoxicity. CONCLUSIONS: The novel cationic ligand (CAE) retains paclitaxel within the phospholipid bilayer and helps in improved drug loading and physical stability. Graphical Abstract ᅟ.

Development and characterization of an organic solvent free, proliposomal formulation of Busulfan using quality by design approach.

Parenteral administration of Busulfan (BU) conquers the bioavailability and biovariability related issues of oral BU by maintaining the plasma drug concentration in therapeutic range with minimal fluctuations thereby significantly reducing the side effects. Busulfex® is the only commercially available parenteral formulation of BU composed of organic solvents N, N-dimethylacetamide and polyethylene glycol 400. Since, BU is highly susceptible to hydrolytic degradation; Busulfex® has poor physical and chemical stability in IV fluids. It is quintessential to develop organic solvent free formulation of BU using parenterally acceptable excipients to enhance its solubility and stability in IV fluids. The Proliposomal formulation of BU was prepared by adsorption-sonicaton method using egg phosphotidylcholine, cholesterol, tween 80 and mannitol. Vesicle size and entrapment efficiency were optimized using 24 full factorial design and characterized by DSC, PXRD and TEM. Optimized formulation spontaneously forms 74.0 ± 1.7 nm sized nanovesicles with 72.9 ± 1.5 % entrapment efficiency. DSC and PXRD studies revealed that BU was present in phospholipid bilayer in amorphized form and TEM images confirmed the multi lamellar vesicular structure. Physicochemical stability of BU was significantly enhanced with proliposomal formulation. In-vivo studies in Sprague Dawley rats showed proliposomal formulation has comparable immunosuppression activity and 110.62 % relative bioavailability as compared to marketed Busulfan formulation i.e. Busulfex®.