Inhalable chitosan-coated nano-assemblies potentiate niclosamide for targeted abrogation of non-small-cell lung cancer through dual modulation of autophagy and apoptosis.

Lung carcinoma, particularly non-small-cell lung cancer (NSCLC), accounts for a significant portion of cancer-related deaths, with a fatality rate of approximately 19 %. Niclosamide (NIC), originally an anthelmintic drug, has attracted attention for its potential in disrupting cancer cells through various intracellular signaling pathways. However, its effectiveness is hampered by limited solubility, reducing its bioavailability. This study investigates the efficacy of NIC against lung cancer using inhalable hybrid nano-assemblies with chitosan-functionalized Poly (ε-caprolactone) (PCL) as a carrier for pulmonary delivery. The evaluation encompasses various aspects such as aerodynamic and physicochemical properties, drug release kinetics, cellular uptake, biocompatibility, cell migration, autophagic flux, and apoptotic cell death in A549 lung cancer cells. Increasing NIC dosage correlates with enhanced inhibition of cell proliferation, showing a dose-dependent profile (approximately 75 % inhibition efficiency at 20 µg/mL of NIC). Optimization of inhaled dosage and efficacy is conducted in a murine model of NNK-induced tumor-bearing lung cancer. Following inhalation, NIC-CS-PCL-NA demonstrates significant lung deposition, retention, and metabolic stability. Inhalable nano-assemblies promote autophagy flux and induce apoptotic cell death. Preclinical trials reveal substantial tumor regression with minimal adverse effects, underscoring the potential of inhalable NIC-based nano-formulation as a potent therapeutic approach for NSCLC, offering effective tumor targeting and killing capabilities.

Targeted delivery of the metastasis-specific tumour homing TMTP1 peptide to non-small-cell lung cancer (NSCLC) using inhalable hybrid nano-assemblies.

Lung cancer is one of the most fatal malignancies, with the highest death rate (∼19%), and the NSCLC type accounts for ∼85% of lung cancers. In the search for new treatments, antimicrobial peptides have received much attention due to their propensity for selective destruction of cancer cells. In the current study, we evaluated the efficacy of the metastasis-specific tumour-homing-TMTP1 peptide against lung cancer using inhalable hybrid nano-assemblies of the PEG-PLGA copolymer as a carrier for pulmonary delivery which was assessed for aerodynamic and physicochemical properties, along with the peptide-release profile, physical stability, cellular uptake and biocompatibility, generation of reactive oxygen species, cell migration, autophagic flux, and apoptotic cell death in A549 lung cancer cells. Optimization of inhaled dose, lung retention, and efficacy studies was conducted to evaluate the formulation in an NNK (nicotine-derived nitrosamine ketone) induced tumour-bearing lung cancer murine model. After inhalation, the formulation with nano-scale physiognomies showed good lung deposition, retention, and metabolic stability. The inhalable nano-assemblies have shown enhanced generation of reactive oxygen species with increased autophagy flux and apoptotic cell death. Pre-clinical animal trials show substantial tumour regression by inhalable TMTP1-based nano-formulation with limited side effects. Our results on metastasis targeting and tumour-homing peptide TMTP1 demonstrate its effective tumour targeting and tumour-killing efficacy and provide a reference for the development of new therapeutics for NSCLC.

Effective cerebral tuberculosis treatment via nose-to-brain transport of anti-TB drugs using mucoadhesive nano-aggregates.

Central nervous system tuberculosis (CNS-TB) is a severe form of extra-pulmonary tuberculosis with high mortality and morbidity rates. The standard treatment regimen for CNS-TB parallels that of pulmonary TB, despite the challenge posed by the blood-brain barrier (BBB), which limits the efficacy of first-line anti-TB drugs (ATDs). Nose-to-brain (N2B) drug delivery offers a promising solution for achieving high ATD concentrations directly at infection sites in the brain while bypassing the BBB. This study aimed to develop chitosan nanoparticles encapsulating ATDs, specifically isoniazid (INH) and rifampicin (RIF). These nanoparticles were further processed into micro-sized chitosan nano-aggregates (NA) via spray drying. Both INH-NA and RIF-NA showed strong mucoadhesion and significantly higher permeation rates across RPMI 2650 cells compared to free ATDs. Intranasal administration of these NAs to TB-infected mice for four weeks resulted in a significant reduction of mycobacterial load by approximately ∼2.86 Log 10 CFU compared to the untreated group. This preclinical data highlights the efficacy of intranasal chitosan nano-aggregates in treating CNS-TB, demonstrating high therapeutic potential, and addressing brain inflammation challenges. To our knowledge, this study is the first to show nasal delivery of ATD nano-formulations for CNS-TB management.

Breaking the Cycle: Matrix Metalloproteinase Inhibitors as an Alternative Approach in Managing Tuberculosis Pathogenesis and Progression.

Mycobacterium tuberculosis (Mtb) has long posed a significant challenge to global public health, resulting in approximately 1.6 million deaths annually. Pulmonary tuberculosis (TB) instigated by Mtb is characterized by extensive lung tissue damage, leading to lesions and dissemination within the tissue matrix. Matrix metalloproteinases (MMPs) exhibit endopeptidase activity, contributing to inflammatory tissue damage and, consequently, morbidity and mortality in TB patients. MMP activities in TB are intricately regulated by various components, including cytokines, chemokines, cell receptors, and growth factors, through intracellular signaling pathways. Primarily, Mtb-infected macrophages induce MMP expression, disrupting the balance between MMPs and tissue inhibitors of metalloproteinases (TIMPs), thereby impairing extracellular matrix (ECM) deposition in the lungs. Recent research underscores the significance of immunomodulatory factors in MMP secretion and granuloma formation during Mtb pathogenesis. Several studies have investigated both the activation and inhibition of MMPs using endogenous MMP inhibitors (i.e., TIMPs) and synthetic inhibitors. However, despite their promising pharmacological potential, few MMP inhibitors have been explored for TB treatment as host-directed therapy. Scientists are exploring novel strategies to enhance TB therapeutic regimens by suppressing MMP activity to mitigate Mtb-associated matrix destruction and reduce TB induced lung inflammation. These strategies include the use of MMP inhibitor molecules alone or in combination with anti-TB drugs. Additionally, there is growing interest in developing novel formulations containing MMP inhibitors or MMP-responsive drug delivery systems to suppress MMPs and release drugs at specific target sites. This review summarizes MMPs' expression and regulation in TB, their role in immune response, and the potential of MMP inhibitors as effective therapeutic targets to alleviate TB immunopathology.

Self-actuating inflammation responsive hydrogel microsphere formulation for controlled drug release in rheumatoid arthritis (RA): Animal trials and study in human fibroblast like synoviocytes (hFLS) of RA patients.

Patients with rheumatoid arthritis (RA) often have one or more painfuljoints despite adequate medicine. Local drug delivery to the synovial cavity bids for high drug concentration with minimal systemic adverse effects. However, anti-RA drugs show short half-lives in inflamed joints after intra-articular delivery. To improve the therapeutic efficacy, it is essential to ensure that a drug is only released from the formulation when it is needed. In this work, we developed an intelligent "Self-actuating" drug delivery system where Disease-modifying anti-rheumatic Drug (DMARD) methotrexate is incorporated within a matrix intended to be injected directly into joints. This formulation has the property to sense the need and release medication only when joints are inflamed in response to inflammatory enzyme Matrix metalloproteinases (MMP). These enzymes are important proteases in RA pathology, and several MMP are present in augmented levels in synovial fluid and tissues. A high level of MMP present in synovial tissues of RA patients would facilitate the release of drugs in response and ascertain controlled drug release. The formulation is designed to be stable within the joint environment, but to dis-assemble in response to inflammation. The synthesized enzyme-responsive methotrexate (Mtx) encapsulated micron-sized polymer-lipid hybrid hydrogel microspheres (Mtx-PLHM) was physiochemically characterized and tested in synovial fluid, Human Fibroblast like synoviocytes (h-FLS) (derived from RA patients) and a rat arthritic animal model. Mtx-PLHM can self-actuate and augment the release of Mtx drug upon contact with either exogenously added MMP or endogenous MMP present in the synovial fluid of patients with RA. The drug release from the prepared formulation is significantly amplified to several folds in the presence of MMP-2 and MMP-9 enzymes. In the rat arthritic model, Mtx-PLHM showed promising therapeutic results with the significant alleviation of RA symptoms through decrease in joint inflammation, swelling, bone erosion, and joint damage examined by X-ray analysis, histopathology and immune-histology. This drug delivery system would be nontoxic as it releases more drug only during the period of exacerbation of inflammation. This will simultaneously protect patients from unwanted side effects when the disease is inactive and lower the need for repeated joint injections.

Clofazimine nanoclusters show high efficacy in experimental TB with amelioration in paradoxical lung inflammation.

The rise of tuberculosis (TB) superbugs has impeded efforts to control this infectious ailment, and new treatment options are few. Paradoxical Inflammation (PI) is another major problem associated with current anti-TB therapy, which can complicate the treatment and leads to clinical worsening of disease despite a decrease in bacterial burden in the lungs. TB infection is generally accompanied by an intense local inflammatory response which may be critical to TB pathogenesis. Clofazimine (CLF), a second-line anti-TB drug, delineated potential anti-mycobacterial effects in-vitro and in-vivo and also demonstrated anti-inflammatory potential in in-vitro experiments. However, clinical implications may be restricted owing to poor solubility and low bioavailability rendering a suboptimal drug concentration in the target organ. To unravel these issues, nanocrystals of CLF (CLF-NC) were prepared using a microfluidizer® technology, which was further processed into micro-sized CLF nano-clusters (CLF-NCLs) by spray drying technique. This particle engineering offers combined advantages of micron- and nano-scale particles where micron-size (∼5 µm) promise optimum aerodynamic parameters for the finest lung deposition, and nano-scale dimensions (∼600 nm) improve the dissolution profile of apparently insoluble clofazimine. An inhalable formulation was evaluated against virulent mycobacterium tuberculosis in in-vitro studies and in mice infected with aerosol TB infection. CLF-NCLs resulted in the significant killing of virulent TB bacteria with a MIC value of ∼0.62 µg/mL, as demonstrated by Resazurin microtiter assay (REMA). In TB-infected mice, inhaled doses of CLF-NCLs equivalent to ∼300 µg and âˆ¼ 600 µg of CLF administered on every alternate day over 30 days significantly reduced the number of bacteria in the lung. With an inhaled dose of ∼600 µg/mice, reduction of mycobacterial colony forming units (CFU) was achieved by ∼1.95 Log10CFU times compared to CLF administered via oral gavage (∼1.18 Log10CFU). Lung histology scoring showed improved pathogenesis and inflammation in infected animals after 30 days of inhalation dosing of CLF-NCLs. The levels of pro-inflammatory mediators, including cytokines, TNF-α & IL-6, and MMP-2 in bronchoalveolar lavage fluid (BAL-F) and lung tissue homogenates, were attenuated after inhalation treatment. These pre-clinical data suggest inhalable CLF-NCLs are well tolerated, show significant anti-TB activity and apparently able to tackle the challenge of paradoxical chronic lung inflammation in murine TB model.

New Generation Smart Drug Delivery Systems for Rheumatoid Arthritis.

Rheumatoid arthritis (RA) is the most common form of the chronic inflammatory autoimmune disease characterized by chronic synovitis, synovial proliferation, and cellular infiltration. Further, it leads to bone erosion, destruction of articular cartilage, intense joint pain, swelling, and a high rate of disability, causing an immense load on human health. If the disease is identified early on, and the patient has continuous and timely treatment, many patients can achieve remission. Although research in RA has made considerable progress, conventional therapies are still the most popular treatment options for most people with RA. But, conventional therapies are hampered by various drawbacks, including higher doses, low solubility and permeability, poor bioavailability, a high level of first-pass metabolism, adaptive treatment tolerance (ATT), and long-term drug use. These drawbacks can result in severe side effects and drug toxicity in patients. Advances in polymer science and the application of nanotechnology in drug delivery systems have provided new possibilities in the treatment of RA by developing new-generation smart drug delivery systems (SDDSs). The shortcomings of non-specific drug distribution and uncontrollable drug release by traditional delivery systems have motivated the creation of next-generation SDDSs. These new smart drug delivery treatment methods have significantly changed the course of RA. Such systems can improve drug delivery by virtue of their multi-functionality and targeting capabilities. The ultimate objective of next-generation SDDSs is to deliver medication at the optimal time with precise dosage and efficiency and specificity to the targeted site (such as cells, tissues, and organs), which can aid patients to adhere better to their therapy. This review highlights and discusses the various next-generation SDDSs along with the conventional treatment options available for RA management.

Taming the Devil: Antimicrobial Peptides for Safer TB Therapeutics.

Tuberculosis (TB) is a highly contagious infection with extensive mortality and morbidity. The rise of TB-superbugs (drug-resistant strains) with the increase of their resistance to conventional antibiotics has prompted a further search for new anti-mycobacterial agents. It is difficult to breach the barriers around TB bacteria, including mycolic cell wall, granuloma, biofilm and mucus, by conventional antibiotics in a short span of time. Hence, there is an essential need for molecules with an unconventional mode of action and structure that can efficiently break the barriers around mycobacterium. Antimicrobial peptides (AMP) are essential components of innate immunity having cationic and amphipathic characteristics. Lines of evidence show that AMPs have good myco-bactericidal and antibiofilm activity against normal as well as antibiotic-resistant TB bacteria. These peptides have shown direct killing of bacteria by membrane lysis and indirect killing by activation of innate immune response in host cells by interacting with the component of the bacterial membrane and intracellular targets through diverse mechanisms. Despite a good anti-mycobacterial activity, some undesirable characteristics are also associated with AMP, including hemolysis, cytotoxicity, susceptibility to proteolysis and poor pharmacokinetic profile, and hence only a few clinical studies have been conducted with these biomolecules. The design of new combinatorial therapies, including AMPs and particulate drug delivery systems, could be new potential alternatives to conventional antibiotics to fight MDR- and XDRTB. This review outlined the array of AMP roles in TB therapy, possible mechanisms of actions, activities, and current advances in pragmatic strategies to improve challenges accompanying the delivery of AMP for tuberculosis therapeutics.

Efficient, enzyme responsive and tumor receptor targeting gelatin nanoparticles decorated with concanavalin-A for site-specific and controlled drug delivery for cancer therapy.

The tumor targeting and stimuli responsiveness behavior of intelligent drug delivery systems imparts effective therapeutic delivery and decreases the toxicity of conventional chemotherapeutic agents in off-target organs. To achieve the receptor targeting and smart drug release, several strategies have been employed to engineer nano-carrier with stimulus sensitivity. In this work, mannose receptor-targeted and matrix metalloproteinase (MMP) responsive gelatin nanoparticles were developed and assessed for its receptor targeting and "on-demand" controlled drug delivery in lung cancer therapeutics. MMPs are protease enzymes and over-expressed in tumorous tissues in all the stages of cancer. The cancer cells also have over-expressed mannose receptors on the cell surface. The surface decoration of gelatin nanoparticles with concanavalin A (con-A) tends to bind with mannose moiety of cell surface glycoproteins which enhances the cancer cell-specific higher uptake of nanoparticles. Gelatin nanoparticles have attracted significant attraction in recent years as a potential drug carrier because of its good biocompatibility and versatile physicochemical properties desirable to deliver the drug. Cisplatin was complexed with the gelatin matrix (CG-NP) to evaluate stimuli responsiveness with the lung cancer cells and its release pattern. In this smart inhalable delivery system, cisplatin loaded gelatin nanoparticles were surface decorated with con-A (CCG-NP). In tumorous cells, con-A coating is expected to enhance mannose receptor-specific cellular internalization of CCG-NP, and subsequently high level of MMP in tumor tissues would help to release cisplatin in response and ensures controlled drug release. The synthesized CCG-NP has shown enzyme triggered drug release and favorable endocytosis after incubation of 12 h compare to uncoated nanoparticles. The efficacy of CCG-NP significantly increased in presence of MMP-2 enzyme in lung cancer cell line A549 cells. It also significantly enhanced reactive oxygen species generation, cell cycle arrest in S and G2/M phase, and apoptosis in cancer cells. Therefore, inhalable CCG-NP promises a pragmatic approach to construct a receptor targeting and an "on-demand" drug delivery system to efficiently deliver the drug at the tumor site only.

Autophagy-Inducing Inhalable Co-crystal Formulation of Niclosamide-Nicotinamide for Lung Cancer Therapy.

Niclosamide (NIC), an anthelminthic drug, is found to be promising in overcoming the problem of various types of drug-resistant cancer. In spite of strong anti-proliferative effect, NIC shows low aqueous solubility, leading to poor bioavailability. To overcome this limitation, and enhance its physicochemical properties and pharmacokinetic profile, we used co-crystallization technique as a promising strategy. In this work, we brought together the crystal and particle engineering at a time using spray drying to enhance physicochemical and aerodynamic properties of co-crystal particle for inhalation purpose. We investigated the formation and evaluation of pharmaceutical co-crystals of niclosamide-nicotinamide (NIC-NCT) prepared by rapid, continuous and scalable spray drying method and compared with conventional solvent evaporation technique. The newly formed co-crystal was evaluated by XRPD, FTIR, Raman spectroscopy and DSC, which showed an indication of formation of H bonds between drug (NIC) and co-former (NCT) as a major binding force in co-crystal development. The particle geometry of co-crystals including spherical shape, size 1-5 µm and aerodynamic properties (ED, 97.1 ± 8.9%; MMAD, 3.61 ± 0.87 µm; FPF, 71.74 ± 6.9% and GSD 1.46) attributes suitable for inhalation. For spray-dried co-crystal systems, an improvement in solubility characteristics (≥ 14.8-fold) was observed, relative to pure drug. To investigate the anti-proliferative activity, NIC-NCT co-crystals were investigated on A549 human lung adenomas cells, which showed a superior cytotoxic activity compared with pure drug. Mechanistically, NIC-NCT co-crystals enhanced autophagic flux in cancer cell which demonstrates autophagy-mediated cell death as shown by confocal microscopy. This technique could help in improving bioavailability of drug, hence reducing the need for high dosages and signifying a novel paradigm for future clinical applications.

Inhalation Delivery of Host Defense Peptides (HDP) using Nano- Formulation Strategies: A Pragmatic Approach for Therapy of Pulmonary Ailments.

Host defense peptides (HDP) are small cationic molecules released by the immune systems of the body, having multidimensional properties including anti-inflammatory, anticancer, antimicrobial and immune-modulatory activity. These molecules gained importance due to their broad-spectrum pharmacological activities, and hence being actively investigated. Presently, respiratory infections represent a major global health problem, and HDP has an enormous potential to be used as an alternative therapeutics against respiratory infections and related inflammatory ailments. Because of their short half-life, protease sensitivity, poor pharmacokinetics, and first-pass metabolism, it is challenging to deliver HDP as such inside the physiological system in a controlled way by conventional delivery systems. Many HDPs are efficacious only at practically high molar-concentrations, which is not convincing for the development of drug regimen due to their intrinsic detrimental effects. To avail the efficacy of HDP in pulmonary diseases, it is essential to deliver an appropriate payload into the targeted site of lungs. Inhalable HDP can be a potentially suitable alternative for various lung disorders including tuberculosis, Cystic fibrosis, Pneumonia, Lung cancer, and others as they are active against resistant microbes and cells and exhibit improved targeting with reduced adverse effects. In this review, we give an overview of the pharmacological efficacy of HDP and deliberate strategies for designing inhalable formulations for enhanced activity and issues related to their clinical implications.

Targeted Pulmonary Delivery of the Green Tea Polyphenol Epigallocatechin Gallate Controls the Growth of Mycobacterium tuberculosis by Enhancing the Autophagy and Suppressing Bacterial Burden.

Growing rates of tuberculosis (TB) superbugs are alarming, which has hampered the progress made to-date to control this infectious disease, and new drug candidates are few. Epigallocatechin gallate (EGCG), a major polyphenolic compound from green tea extract, shows powerful efficacy against TB bacteria in in vitro studies. However, the therapeutic efficacy of the molecule is limited due to poor pharmacokinetics and low bioavailability following oral administration. Aiming to improve the treatment outcomes of EGCG therapy, we investigated whether encapsulation and pulmonary delivery of the molecule would allow the direct targeting of the site of infection without compromising the activity. Microencapsulation of EGCG was realized by scalable spray-freeze-drying (SFD) technology, forming free-flowing micrometer-sized microspheres (epigallocatechin-3-gallate-loaded trehalose microspheres, EGCG-t-MS) of trehalose sugar. These porous microspheres exhibited appropriate aerodynamic parameters and high encapsulation efficiencies. In vitro studies demonstrated that EGCG-t-MS exhibited dose- and time-dependent killing of TB bacteria inside mouse macrophages by cellular mechanisms of lysosome acidification and autophagy induction. In a preclinical study on TB-infected Balb/c mice model (4 weeks of infection), we demonstrate that the microencapsulated EGCG, administered 5 days/week for 6 weeks by pulmonary delivery, showed exceptional efficacy compared to oral treatment of free drug. This treatment approach exhibited therapeutic outcomes by resolution of inflammation in the infected lungs and significant reduction (P < 0.05) in bacterial burden (up to ∼2.54 Log10 CFU) compared to untreated control and orally treated mice groups. No pathological granulomas, lesions, and inflammation were observed in the histopathological investigation, compared to untreated controls. The encouraging results of the study may pave the avenues for future use of EGCG in TB therapeutics by targeted pulmonary delivery and lead to its translational success.

Matrix Metalloproteinase-Responsive Mesoporous Silica Nanoparticles Cloaked with Cleavable Protein for "Self-Actuating" On-Demand Controlled Drug Delivery for Cancer Therapy.

The tumour site-specific stimulus responsiveness of smart drug delivery systems gives a unique system for effective therapeutic delivery with reduced toxic effects of conventional chemotherapeutic drugs. In this work, matrix metalloproteinase-2 (MMP-2)-responsive mesoporous silica nanoparticles (MSNs) were synthesized and assessed for "self-actuating" on-demand controlled drug delivery for cancer therapy. MMPs are members of protease enzymes that are generally overexpressed in cancerous tissues in all stages of cancer. MSNs have attracted significant consideration as a potential delivery system because of their robust and versatile physicochemical properties suitable to deliver the therapeutic payload. Cisplatin (Cis) was used as a model drug, which was incorporated into MSNs to evaluate targeting of lung cancer cells and their release kinetics. In this delivery system, collagen was coated on the surface of Cis-loaded MSNs (Cis-MSN) to form a capping layer, resulting in collagen-coated MSNs (Cis-col-MSN). Under normal cell conditions, a collagen-capping coat efficiently forbids the release of Cis molecules from Cis-col-MSN. The tumor microenvironment would lead to augmented drug release because of the uncapping of collagen from MSN pores due to the presence of overexpressed MMP-2 enzyme and the ensuing controlled drug release. MMP-responsive experiments have shown augmented enzyme triggered drug release. The cellular uptake and cytocompatibility studies in A549 adenocarcinomic lung cancer cell lines demonstrated that this nanocarrier could be efficiently endocytosed in 24 h and have shown favorable biocompatibility with the cells. Cytotoxicity results of Cis-col-MSN demonstrated dose-dependent toxicity. The efficacy of the Cis-col-MSN significantly enhanced with the supplementation of MMP-2 enzyme with increasing concentrations in the cell culture milieu. The efficacy of formulation was attributed to significantly enhance reactive oxygen species, cell cycle arrest, and apoptosis. It is expected that Cis-col-MSN promises a pragmatic approach to constructing an "on-demand" smart drug delivery system to deliver a therapeutic payload at the tumor site only.

Alginate Microspheres Elicit Innate M1-Inflammatory Response in Macrophages Leading to Bacillary Killing.

Particulate drug delivery systems (PDDS) have been broadly explored as platforms for delivery of drugs, enzymes, cells, and vaccines for pharmaceutical applications. Studies suggest that microspheres (MS) can stimulate innate immune cells even without a drug payload; however, less is known regarding how they impact host cells in dealing with the bacillary infection. We examined the role of drug-free inhalable alginate microspheres (A-MS) on phagocytosis efficiency and subsequent immune cell activation in Escherichia coli-infected THP-1-derived macrophages. Alginate particles have been widely investigated as carriers for prolonged delivery of bioactive (i.e., drugs, diagnostics, and vaccines). A-MS were fabricated by industry scalable spray-congealing process using divalent cation-induced gelification. E. coli-infected macrophages (multiplicity of infection (MOI 1:10) were treated with drug-free A-MS, where we found a consistent moderate reduction in bacillary viability. Particles were more efficiently and rapidly phagocytized by infected macrophages as compared with normal macrophage cells. Subsequently, A-MS induced markers of M1 macrophage responses and stimulated the processing and secretion of pro-inflammatory cytokines (IL-6, IL-12). It also notably augmented the generation of reactive oxygen species (ROS) and nitric oxide (NO) in infected cells. Results illustrate that, the blank A-MS (without a drug payload) able to moderately check the growth of intracellular E. coli (without significant cytotoxicity) by modulating the M1 inflammatory response by host cells. This "added value" can be utilized in the design and development of therapeutic system with the additional advantage of immune-modulatory activity, in addition to serving as a drug carrier.

Mycobactericidal activity of some micro-encapsulated synthetic Host Defense Peptides (HDP) by expediting the permeation of antibiotic: A new paradigm of drug delivery for tuberculosis.

Resistance to anti-Tuberculosis (anti-TB) drugs is primarily due to unique intrinsic resistance mechanisms that mycobacterium possess. The most important determinant of resistance is a peculiar hydrophobic and multi-layered mycobacterial cell-wall structure with mycolic-acid and wax-D, which restricts permeability of both hydrophobic and hydrophilic drugs into bacteria. In this study, it was supposed that Host Defense peptides (HDP) which are known to permeabilize bacterial membranes may, therefore, help anti-TB antibiotics to target internal sites in bacteria. To test this hypothesis, we examined the effect of suboptimal concentration (10 µg/ml) of selected microencapsulated-HDP (Ub2-MS, K4-MS, and Aurein1.2-MS) with a standard anti-TB drug (Isoniazid, INH, 3 µg/ml). We also examined the combined effect of different concentrations of HDP-MS with a suboptimal concentration of anti-TB drug (INH, 1.5 µg/ml) which showed additive efficacy. A number of cationic HDP were encapsulated in inhalable microspheres (HDP-MS) and characterized for physicochemical and aerodynamic properties. These peptides were further evaluated for molecular mass by MALDI-TOF and random coil in its secondary structure as determined by circular dichroism. The anti-mycobacterial kinetics of selected HDP-MS (Ub2-MS, K4-MS, and Aurein1.2-MS) was evaluated against virulent Mycobacterium tuberculosis (Mtb), both alone and in conjunction with anti-TB drug (INH). HDP-MS exhibited up to ∼3.02 and ∼3.41-log decrease in CFU as compared to blank-MS (drug free) and untreated control group in 96 h. The combination of HDP-MS with a suboptimal concentration of INH (1.5 µg/ml) showed superior antibiotic activity against Mtb. Our findings show that the enhanced efficacy is due to augmentation of membrane permeation by HDP which expedited the entry of TB drug into apparently the impermeant mycobacterial membrane which further enhances the effective efficacy of the drug. This phenomenon can reduce the need for high dosages and represents a novel paradigm for potential clinical applications.

Heparin-Encapsulated Metered-Dose Topical "Nano-Spray Gel" Liposomal Formulation Ensures Rapid On-Site Management of Frostbite Injury by Inflammatory Cytokines Scavenging.

The critical time window between the incidence of frostbite injury and the initiation of treatment in remote snowbound areas is a determining factor for an effective therapeutic response. It is an emergency condition and challenging to treat due to the poor vascularity of affected body parts, and it requires immediate action. In addition to cold trauma-induced tissue damage, the inflammatory mediators majorly contribute to pathologic aggravations. We have designed and evaluated a topical "nano-spray gel (NSG)" formulation, which is based on a combination of liposomal heparin sodium (Hp) and ibuprofen (Ibu) for rapid relief of frostbite injury in extremely low temperatures. The scientific literature suggests that heparin is associated with rapid endothelial cell repair, normalizing blood circulation in capillaries, and has a potential role in wound healing. Hp-containing liposomes were prepared by the extruder method, which suitably formulated an ibuprofen-containing gel to obtain a nano-Spray formulation (HLp-Ibu-NSG) applicable for topical delivery. A single spray puff of the formulation delivers ∼154 mg of the gel, which corresponds to ∼205 U of heparin. In this study, heparin liposomes exhibited significant healing of wound in vitro (scratch assay, fibroblast cells) and in vivo (wound healing in Sprague Dawley rats) at a low dose. In the rat model of frostbite injury, the HLp-Ibu-NSG formulation demonstrated significant reduction in the wound area (up to ∼96%) and improvement of histopathology in 14 days as compared to the control groups. No edema and erythema were detected post-treatment of HLp-Ibu-NSG in the affected area. The underlying mechanism was delineated as a modulation of the inflammatory cytokine (IL-6, TNF-α, IL-10, IL-4) mediators at the wound site and blood circulation to foster frostbite healing. Future clinical studies on the nano-spray gel are required to evaluate its efficacy for the treatment of frostbite symptoms. The instant on-site application of this formulation might be helpful in saving extremities of soldiers, mountaineers, and pilgrims having frostbite.

Nano-encapsulated HHC10 host defense peptide (HDP) reduces the growth of Escherichia coli via multimodal mechanisms.

The eradication of several pathogenic drug resistant "Superbug" such as Escherichia coli became difficult especially in chronic infections using existing antibiotics due to the emergence of antibiotic resistance. Owing to their unique antibacterial properties, host defense peptides (HDP) have gained significant attention to combat colonization of bacteria. This study aims designing delivery systems for HHC10 peptide to target bacteria inside the cells might be a promising approach by protecting from degradation, controlling the release, enhancing the susceptibility of target microbes and improving bioavailability. Nano-formulated HHC10 was evaluated for its efficacy (CFU assay) and possible mechanism of action (membrane interaction and apoptosis) against E. coli. Dose-dependent inhibition of E. coli growth is observed for nano-encapsulated and bare HHC10 and encapsulated form remain non-toxic to macrophage mouse cells (RAW264.6) up to 20 µM. Mechanistic analyses using transmission electron microscopy and flow cytometry techniques revealed that bactericidal activity of HHC10-NP progresses via a multimodal mechanism of bacterial cell death by cell-membrane lysis on direct interaction with bacteria while through induction of the apoptotic death pathway inside the host cells. These results offer an insight on future strategies for the development and application of antimicrobial peptides as antibacterial alternatives. Controlled delivery of HHC10 peptide from PLGA-NP kills bacteria by two different mechanism: (i) direct killing: HHC10 disintegrate the cell membrane of bacteria by electrostatic interactions and (ii) indirect killing: induction of apoptosis in bacteria infect cells.

Lysosomal targeting strategies for design and delivery of bioactive for therapeutic interventions.

Lysosomes are of particular interest for the design and delivery of pH-dependent pro-drugs, enhancing selectivity and developing strategies to inhibit drug degradation inside the cells. There is great potential to bring intracellular drug delivery and distribution using nanotherapeutic approaches to target lysosomes for therapeutic interventions. Lysosomal targeting strategies involve two contrasting facets. One aspect is to directly target therapeutics to the lysosome through receptor-mediated endocytosis and the other facet involves strategies, which ensure escape from the lysosome in order to prevent their degradation, so that therapeutics may remain intact and available in the cytosol for their further action. It provides a unique opportunity to explore novel treatment strategies and design future drugs for the effective treatment of lysosome-related diseases especially lysosomal storage disorders (LSD), cancer, inflammatory, neurodegenerative conditions (Parkinson's, Alzheimer's and Huntington's diseases) and autoimmune diseases. In this review, we illustrate the fundamentals of membrane trafficking, subcellular organisation, strategies to target lysosomes and its implications for the advance design of efficient drug delivery vectors for safe and effective therapies.

Curcumin-loaded chitosan-cholesterol micelles: evaluation in monolayers and 3D cancer spheroid model.

AIM: To improve the bioavailability and anticancer potential of curcumin by using a cholesterol-conjugated chitosan micelle. Methods & methods: Cholesterol was conjugated to chitosan (15 kDa) to form self-assembled micelles, which loaded curcumin. Physicochemical characterization and formulation optimization of the drug-loaded micelles (curcumin-loaded chitosan-cholesterol micelles [C-CCM]) were performed. In vitro cellular uptake and viability of C-CCM were investigated in melanoma and breast cancer cell lines. The antitumor efficacy was evaluated in 3D lung cancer spheroid model. RESULTS & CONCLUSION: The optimized C-CCM had size of approximately 162 nm with loading efficiency of approximately 36%. C-CCM was taken up efficiently by the cells, and it reduced cancer cell viability significantly compared with free curcumin. C-CCM enhanced the antitumor efficacy in spheroids, suggesting that C-CCM could be used as an effective chemotherapy in cancer.