Remote loading in liposome: a review of current strategies and recent developments.

Liposomes have gained prominence as nanocarriers in drug delivery, and the number of products in the market is increasing steadily, particularly in cancer therapeutics. Remote loading of drugs in liposomes is a significant step in the translation and commercialization of the first liposomal product. Low drug loading and drug leakage from liposomes is a translational hurdle that was effectively circumvented by the remote loading process. Remote loading or active loading could load nearly 100% of the drug, which was not possible with the passive loading procedure. A major drawback of conventional remote loading is that only a very small percentage of the drugs are amenable to this method. Therefore, methods for drug loading are still a problem for several drugs. The loading of multiple drugs in liposomes to improve the efficacy and safety of nanomedicine has gained prominence recently with the introduction of a marketed formulation (Vyxeos) that improves overall survival in acute myeloid leukemia. Different strategies for modifying the remote loading process to overcome the drawbacks of the conventional method are discussed here. The review aims to discuss the latest developments in remote loading technology and its implications in liposomal drug delivery.

Liposomal drug delivery to the lungs: a post covid-19 scenario.

High local delivery of anti-infectives to the lungs is required for activity against infections of the lungs. The present pandemic has highlighted the potential of pulmonary delivery of anti-infective agents as a viable option for infections like Covid-19, which specifically causes lung infections and mortality. To prevent infections of such type and scale in the future, target-specific delivery of drugs to the pulmonary region is a high-priority area in the field of drug delivery. The suboptimal effect of oral delivery of anti-infective drugs to the lungs due to the poor biopharmaceutical property of the drugs makes this delivery route very promising for respiratory infections. Liposomes have been used as an effective delivery system for drugs due to their biocompatible and biodegradable nature, which can be used effectively for target-specific drug delivery to the lungs. In the present review, we focus on the use of liposomal drug delivery of anti-infectives for the acute management of respiratory infections in the wake of Covid-19 infection.

Enhanced skin penetration of Finasteride loaded DMSO-liposomes for the treatment of androgenic alopecia: comparison with conventional liposomes.

Long-term treatment with finasteride (FIN) for androgenic alopecia is restricted due to its systemic side effects. To address this problem, DMSO-modified liposomes were prepared in the present study to improve the topical delivery of FIN. DMSO-liposomes were prepared by a modification of the ethanol injection method. It was hypothesized that the permeation-enhancing property of DMSO could promote drug delivery to deeper skin layer where hair follicles are present. Liposomes were optimized by quality by design (QbD) approach and biologically evaluated in a rat model of testosterone-induced alopecia. Optimized DMSO-liposomes were spherical and had mean vesicle size, zeta potential, and entrapment efficiency of 330.1 ± 1.5, -14.52 ± 1.32, and 59.02 ± 1.12%, respectively. Biological evaluation on testosterone-induced alopecia and skin histology shows that follicular density and anagen/telogen (A/T) ratio were increased in rats treated with DMSO-liposomes as compared to FIN-liposomes without DMSO and an alcoholic solution of FIN applied topically. DMSO-liposomes could be promising skin delivery vehicles for FIN or similar drugs.

Permeation enhancer nanovesicles mediated topical delivery of curcumin for the treatment of hyperpigmentation.

The main aim of the present study was to develop curcumin (CUR) loaded permeation enhancer-lipid vesicles for the treatment of hyperpigmentation. Hyperpigmentation is an acquired skin disorder characterized by uneven skin coloration, mainly in the regions of the facial skin, affecting millions of people worldwide. It often occurs in visible areas, hence causing significant negative psychological and social impacts. In the present study, curcumin-loaded permeation enhancer nanovesicles (PE-NVs) were developed by modified ethanol injection method and dimethyl sulfoxide was added as a penetration enhancer. PE-NVs were subjected to various physicochemical characterizations and drug permeation studies across the skin. The PE-NVs were tested for their efficacy in a sunlight-induced hyperpigmented rabbit skin model. Topical application of PE-NVs reduced symptoms of hyperpigmentation as compared with CUR methanolic solution because of higher accumulation because of better permeation into skin layers. Histopathological studies also confirmed the effectiveness of PE-NVs, since they reduced hyperpigmentation-induced lesions. Results confirmed that PE-NVs is a potential drug delivery system for topical administration drugs to treat skin-associated inflammatory disorders.

A mechanistic review on the dissolution phase behavior and supersaturation stabilization of amorphous solid dispersions.

Amorphous solid dispersion (ASD) technology is an attractive formulation approach for poorly soluble drugs because of the supersaturated state acquired during its dissolution. The high thermodynamic activity of the supersaturated state of the drug is also a driver for the enhanced absorptive flux across a membrane. However, this advantage can easily be lost due to the inherent instability of supersaturation, causing drug precipitation. Stabilizing the supersaturated state during the dissolution of ASD for the relevant absorption time frame is a challenging area in formulation research. Stabilizing the supersaturated state by using polymeric excipients and understanding the phase behavior of drugs during dissolution are required for the optimal performance of ASD formulations. A number of confounding kinetic, formulation and physiological factors can influence the evolution of supersaturation and phase changes during dissolution of ASDs. The review highlights the complex nature of dissolution of ASDs and the need of biorelevant dissolution for proper risk assessment and optimizing formulation development.

Role of Permeability on the Biopredictive Dissolution of Amorphous Solid Dispersions.

An ideal dissolution test for amorphous solid dispersions (ASDs) should reflect physicochemical, physiological, and hydrodynamic conditions which accurately represent in vivo dissolution. However, this is confounded by the evolution of different molecular and colloidal species during dissolution, generating a supersaturated state of the drug. The supersaturated state of a drug is thermodynamically unstable which drives the process of precipitation resulting in a loss of solubility advantage. Maintaining a supersaturated state of the drug with the help of precipitation inhibiting excipients is a key component in the design of ASDs. Therefore, a biopredictive dissolution test is critical for proper risk assessment during the development of an optimal ASD formulation. One of the overlooked components of biopredictive dissolution is the role of drug permeability. The kinetic changes in the phase behavior of a drug during dissolution of ASDs are influenced by drug permeability across a membrane. Conventionally, drug dissolution and permeation are analyzed separately although they occur simultaneously in vivo. The kinetic phase changes occurring during dissolution of ASDs can influence the thermodynamic activity and membrane flux of a drug. The present review evaluates the feasibility, predictability, and practicability of permeability/dissolution for the optimal development and risk assessment of ASD formulations.

Targeting metabolic syndrome with phytochemicals: Focus on the role of molecular chaperones and hormesis in drug discovery.

Adaptive cellular stress response confers stress tolerance against inflammatory and metabolic disorders. In response to metabolic stress, the key mediator of cellular adaptation and tolerance is a class of molecules called the molecular chaperones (MCs). MCs are highly conserved molecules that play critical role in maintaining protein stability and functionality. Hormesis in this context is a unique adaptation mechanism where a low dose of a stressor (which is toxic at high dose) confers a stress-resistant adaptive cellular phenotype. Hormesis can be observed at different level of biological organization at various measurable endpoints. The MCs are believed to play a key role in adaptation during hormesis. Several phytochemicals are known for their hormetic response and are called phytochemical hormetins. The role of phytochemical-mediated hormesis on the adaptive cellular processes is proposed as a potential therapeutic approach to target inflammation associated with metabolic syndrome. However, the screening of phytochemical hormetins would require a paradigm shift in the methods currently used in drug discovery.

Unmet technological demands in orodispersible films for age-appropriate paediatric drug delivery.

Age-appropriateness of a formulation is the ability to deliver variable but accurate doses to the paediatric population in a safe and acceptable manner to improve medical adherence and reduce medication errors. Paediatric drug delivery is a challenging area of formulation research due to the existing gap in knowledge. This includes the unknown safety of excipients in the paediatric population, the need for an age-appropriate formulation, the lack of an effective taste-masking method and the lack of paediatric pharmacokinetic data and patient acceptability. It is equally important to establish methods for predicting the biopharmaceutical performance of a paediatric formulation as a function of age. Overcoming the challenges of existing technologies and providing custom-made solutions for the development of age-appropriate formulation is, therefore, a daunting task. Orodispersible films (ODF) are promising as age-appropriate formulations, an unmet need in paediatric drug delivery. New technological improvements in taste masking, improving solubility and rate of dissolution of insoluble drugs, the flexibility of dosing and extemporaneous preparation of these films in a hospital good manufacturing practises (GMP) setup using 3D printing can increase its acceptance among clinicians, patients and caregivers. The current review discusses the problems and possibilities in ODF technology to address the outstanding issues of age-appropriateness, which is the hallmark of patient acceptance and medical adherence in paediatrics.

Comprehensive profiling of traditional herbomineral formulation Manasamitra vatakam in rat brain following oral administration and in-silico screening of the identified compound for anti-Alzheimer's activity.

ETHNOPHARMACOLOGICAL RELEVANCE: Multi-targeted drug therapy has received substantial attention for the treatment of diseases of multifactorial origin, such as neurodegenerative diseases. Manasamitra vatakam (MMV) is a traditional Ayurvedic formulation used to improve cognitive impairment and mental illness. Here we have used a unique method for leveraging the barrier properties of the intestinal and blood-brain barrier (BBB) to screen and identify the bioactive molecules against Alzheimer's disease (AD). The current method exemplifies a facile method to expedite drug discovery from traditional formulations. AIM OF THE STUDY: The present study aimed to identify the phytoconstituents of MMV that reach the brain tissue and to predict major bioactive constituents by computational docking studies. MATERIALS AND METHODS: After oral administration of the formulation, brain samples from male Sprague Dawley rats were collected at different time intervals and analyzed by liquid chromatography-mass spectrometry (LC-MS) to identify the phytoconstituents. In silico molecular docking studies were carried out to analyze the binding affinity of the compounds to the target proteins of AD using Schrodinger Maestro. The molecular dynamic studies were carried out for all the docked complexes having higher docking scores. RESULTS: 34 phytoconstituents were identified by LC-MS analysis of brain homogenates. In the in silico docking study, the phytoconstituents chrysin, convolvin, rutin, galangin, palmatoside G, isoliquiritigenin, quercetin, and naringenin showed higher docking score against the target proteins of AD. These compounds may serve as the primary bioactive compounds responsible for the neuroprotective activity of the herbal formulation. Furthermore, molecular dynamic studies indicated that the galangin-acetylcholinesterase enzyme complex has the highest stability among these eight compounds. CONCLUSION: The study, together with previous in vivo and in vitro efficacy results, suggests that BBB-permeable compounds with high binding affinities for the target proteins of AD might be responsible for the effectiveness of MMV against AD.

Physiological relevance of in-vitro cell-nanoparticle interaction studies as a predictive tool in cancer nanomedicine research.

Cell uptake study is a routine experiment used as a surrogate to predict in vivo response in cancer nanomedicine research. Cell culture conditions should be designed in such a way that it emulates 'real' physiological conditions and avoid artefacts. It is critical to dissect the steps involved in cellular uptake to understand the physical, chemical, and biological factors responsible for particle internalization. The two-dimensional model (2D) of cell culture is overly simplistic to mimic the complexity of cancer tissues that exist in vivo. It cannot simulate the critical tissue-specific properties like cell-cell interaction and cell-extracellular matrix (ECM) interaction and its influences on the temporal and spatial distribution of nanoparticles (NPs). The three dimensional model organization of heterogenous cancer and normal cells with the ECM acts as a formidable barrier to NP penetration and cellular uptake. The three dimensional cell culture (3D) technology is a breakthrough in this direction that can mimic the barrier properties of the tumor microenvironment (TME). Herein, we discuss the physiological factors that should be considered to bridge the translational gap between in and vitro cell culture studies and in-vivo studies in cancer nanomedicine.

Therapeutic Approaches for Intravascular Microthrombi-induced Acute Respiratory Distress Syndrome (ARDS) in COVID-19 Infection.

The COVID-19 pandemic has overwhelmed our health care capacity in an unprecedented way due to the sheer number of critically infected patients admitted to hospitals during the last two years. Endothelial injury is seen as one of the central hallmarks of COVID-19 infection that is the starting point in the generation of microthrombi and sepsis eventually leading to acute respiratory distress syndrome (ARDs) and multi-organ failure. The dramatic fall in lung function during ARDs is attributed to the microthrombi-induced coagulopathy primed by a hyperactive immune system. Due to the lack of effective antiviral agents, the line of treatment is limited to the management of two key risk factors i.e., immune activation and coagulopathy. In the present review, we describe the mechanistic role, therapeutic targets, and opportunities to control immune activation and coagulopathy during the pathogenesis of COVID-19-induced ARDs.

Revisiting the outstanding questions in cancer nanomedicine with a future outlook.

The field of cancer nanomedicine has been fueled by the expectation of mitigating the inefficiencies and life-threatening side effects of conventional chemotherapy. Nanomedicine proposes to utilize the unique nanoscale properties of nanoparticles to address the most pressing questions in cancer treatment and diagnosis. The approval of nano-based products in the 1990s inspired scientific explorations in this direction. However, despite significant progress in the understanding of nanoscale properties, there are only very few success stories in terms of substantial increase in clinical efficacy and overall patient survival. All existing paradigms such as the concept of enhanced permeability and retention (EPR), the stealth effect and immunocompatibility of nanomedicine have been questioned in recent times. In this review we critically examine impediments posed by biological factors to the clinical success of nanomedicine. We put forth current observations on critical outstanding questions in nanomedicine. We also provide the promising side of cancer nanomedicine as we move forward in nanomedicine research. This would provide a future direction for research in nanomedicine and inspire ongoing investigations.

Combined adjuvant-delivery system for new generation vaccine antigens: alliance has its own advantage.

Vaccines are a significant historical accomplishment in medical science due to its significance in saving millions of lives around the world with a manifold decline in disease burden and health expenditure. Since last decade, extensive efforts in vaccine design based on rational and experimental set up prompts the acknowledgement of several protective antigens; however, the utilization of those antigens as intense safe vaccines is still far beyond their discovery. The advancement of accomplished vaccines will need the combination of numerous strategies. In this way, the vaccine can be capable of provoking an abundant and effective immunization to compete against antigens with minimal/no adverse effects on recipients. First, stable and striking enough on the pharmaceutical point of view. Second, application of rational and realistic approaches to select an appropriate combination of adjuvant, antigen and delivery vehicle in the suitable formulation. Different vaccine adjuvants-delivery system combinations were discussed here elaborately, which are approved/licensed for human vaccines and are in various phases of clinical trials and pre-clinical trials. Challenges/limitations associated with vaccine designing and parameters to be considered, approaches to be applied, using available adjuvants are also detailed.

In-vitro in-vivo correlation (IVIVC) in nanomedicine: Is protein corona the missing link?

One of the unmet challenges in nanotechnology is to understand and establish the relationship between physicochemical properties of nanoparticles (NPs) and its biological interactions (bio-nano interactions). However, we are still far from assessing the biofate of NPs in a clear and unquestionable manner. Recent developments in the area of bio-nano interface and the understanding of protein corona (PC) has brought new insight in predicting biological interactions of NPs. PC refers to the spontaneous formation of an adsorbed layer of biomolecules on the surface of NPs in a biological environment. PC formation involves the spatiotemporal interplay of an intricate network of biological, environmental and particle characteristics. NPs with its PC can be viewed as a biological entity, which interacts with cells and barriers in a biological system. Recent studies on the bio-nano interface have revealed biological signatures that participate in cellular and physiological bioprocesses and control the biofate and toxicity of NPs. The ability of in-vitro derived parameters to forecast in-vivo consequences by developing a mathematical model forms the basis of in-vitro in-vivo correlation (IVIVC). Understanding the effect of bio-nano interactions on the biological consequences of NPs at the cellular and physiological level can have a direct impact on the translation of future nanomedicines and can lead to the ultimate goal of developing a mathematical IVIVC model. The review summarizes the emerging paradigms in the field of bio-nano-interface which clearly suggests an urgent need to revisit existing protocols in nanotechnology for defining the physicochemical correlates of bio-nano interactions.

Nanoparticle-based immunopotentiation via tetanus toxoid-loaded gelatin and aminated gelatin nanoparticles.

Gelatin nanoparticles (GNPs) and aminated gelatin nanoparticles (AGNPs) were prepared and used as an adjuvant to improve the delivery of tetanus toxoid (TT). Nanoparticles were characterized in vitro for their size, shape, entrapment, and release. TT-FITC conjugate was used to determine entrapment and release from nanoparticles. The immune-stimulating activity was studied by measuring anti-TT IgG, IgG1, and IgG2a isotype and cytokine responses following subcutaneous (s.c) injection of nanoparticles in BALB/c mice and was compared with alum-TT vaccine. Gelatin and aminated gelatin (AG) specific IgG response was also determined. Both GNPs and AGNPs demonstrated comparable IgG response and a significantly higher (p < 0.05) cytokine response (IL-2 and IFN γ) as compared to alum-TT vaccine. Nanoparticulate formulations elicited both Th1 and Th2 responses and induced negligible undesirable immunogenicity against the carrier, as demonstrated by lower level of gelatin and AG-specific IgG response as compared to control.

Comparison of humoral and cell-mediated immune responses to cationic PLGA microspheres containing recombinant hepatitis B antigen.

Presently available marketed alum adsorbed hepatitis B vaccine used for prophylactic immunization, can effectively elicit humoral immunity but is poor inducer of cell-mediated immunity (CMI). Besides, conventional alum-adjuvant vaccines require multiple injections to achieve long-lasting protective immune responses. Therefore, as a result of insufficient immunization, infections are still the leading killer among diseases. The present investigation was therefore, aimed at developing "single-shot" HBsAg adsorbed microspheres of poly (DL)-lactide-co-glycolide (PLGA) (L/G 50:50 and 75:25) and their capability to stimulate the cell mediated immune response against hepatitis B surface antigen. These microspheres were characterized in vitro for their size, shape polydispersity index, percentage HBsAg adsorption efficiency and in vitro release profile. The immune-stimulating activities were also studied following subcutaneous injection of HBsAg adsorbed PLGA microspheres (single-dose on day 0) and compared with alum adsorbed vaccines (two-doses on 0 and 28 days) in Balb/c mice. Specific cell-mediated immune responses such as lymphocyte transformation assay (stimulation-index) including release of interferon-gamma (IFN-γ), interleukin-2 (IL-2) and nitric-oxide were determined. Cellular responses in case of alum adsorb HBsAg vaccine was very low. These studies demonstrate the potential of cationic polymeric microspheres based vaccine in stimulating cell mediated immune response along with humoral response against hepatitis B.

Carbohydrate modified ultrafine ceramic nanoparticles for allergen immunotherapy.

The uses of drug-delivery systems in allergen specific immunotherapy appear to be a promising approach due to their ability to act as adjuvants, transport the allergens to immune-competent cells and tissues and reduce the number of administrations. The aim of this work was to evaluate the carbohydrate modified ultrafine ceramic core based nanoparticles (aquasomes) as adjuvant/delivery vehicle in specific immunotherapy using ovalbumin (OVA) as an allergen model. Prepared nanoparticles were characterized for size, shape, zeta potential, antigen integrity, surface adsorption efficiency and in vitro release. The humoral and cellular-induced immune responses generated by OVA adsorbed aquasomes were studied by two intradermal immunizations in BALB/c mice. OVA sensitized mice were treated with OVA adsorbed aquasomes and OVA adsorbed aluminum hydroxide following established protocol. Fifteen days after therapy, animals were challenged with OVA and different signs of anaphylactic shock were evaluated. Developed aquasomes possessed a negative zeta potential (-11.3 mV) and an average size of 47 nm with OVA adsorption efficiency of ~60.2 µg mg(-1) of hydroxyapatite core. In vivo immune response after two intradermal injections with OVA adsorbed aquasomes resulted in a mixed Th1/Th2-type immune response. OVA-sensitized mice model, treatment with OVA adsorbed aquasomes elicited lower levels of IgE (p<0.05), serum histamine and higher survival rate in comparison with alum adsorbed OVA. Symptoms of anaphylactic shock in OVA aquasome-treated mice were weaker than the one induced in the alum adsorbed OVA group. Results from this study demonstrate the valuable use of aquasomes in allergen immunotherapy.

Humoral and cell-mediated immune-responses after administration of a single-shot recombinant hepatitis B surface antigen vaccine formulated with cationic poly(l-lactide) microspheres.

The present investigations were aimed to compare the humoral and cell-mediated immune responses between recombinant hepatitis B surface antigens (HBsAg) adsorbed L-PLA microspheres (Ms) vaccine (single-shot) and marketed alum-HBsAg vaccine (two-doses). The blank cationic (cetyltrimethyammoniumbromide) microspheres were prepared by the double emulsion (w/o/w) solvent evaporation technique. The HBsAg was adsorbed onto the surface of blank cationic microspheres. These microspheres were characterized in vitro for their size, shape, adsorption-efficiency, in-process stability, and HBsAg release studies. Specific humoral immune responses (IgM and IgG) and cell-mediated immune responses (cellular-proliferation) assay including release of interferon-gamma (IFN-gamma), interleukin-2 (IL-2), and nitric oxide (NO) from host's cells stimulated with HBsAg or lipopolysaccharide (LPS)/ concanavalin A (con A) in-vitro were determined. Based on these findings, it was concluded that the single injection (using subcutaneous-route) of the polymeric microspheres produced better immune response (both humoral and cell-mediated) than two injections of a conventional alum-HBsAg vaccine. These data demonstrate high potential of polymeric microspheres for their use as a carrier adjuvant for hepatitis B vaccine.

Development and characterization of lectin-functionalized vesicular constructs bearing amphotericin B for bio-film targeting.

The long-term goal of this work will be to develop a topical formulation for oropharyngeal candidosis. Liposomes were prepared by the vesicle extrusion technique from mixtures of dipalmitoylphosphatidylcholine, cholesterol, and stearylamine incorporating a reactive phospholipid, the m-maleimidobenzoyl-N-hydroxysuccinimide ester derivative of dipalmitoylphosphatidylethanolamine, which was conjugated with the N-succinimidyl-S-acetylthioacetate derivatives of succinyl concanavalin A. Morphology of liposomes was studied by transmission electron microscopy and bio-film architecture using fluorescence microscopy. Lectinized vesicles were put into contact with bovine submaxillary mucin, to determine the in vitro activity and specificity. Targeting study was performed using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. The liposomes were found to have a diameter in the range of 360-450 and 390-510 nm for uncoated and coated formulations, respectively. The MTT assay showed a strong association of lectin-bearing liposomes with candidal bio-film. The results suggest that surface-modified liposomes can effectively target candidal bio-film in vitro.