"Y-type” PEG modified liposomes could eliminate the accelerated blood clearance (ABC) phenomenon and improved tumor therapy

Poly(ethylene glycol) (PEG) is widely applied to decorate nanocarriers due to its "long circulation” characteristics. However, the applications of linear PEG-modified nanocarriers have been hindered by severe adverse effects due to the accelerated blood clearance (ABC) phenomenon. It was universally known that anti-PEG antibodies (APAs) were main culprits in ABC phenomenon which induced the significant change in pharmacokinetics, biological distributions of the second injection and triggered complement activation-related pseudoallergies (CARPA). Recent studies have illustrated that APAs triggered the ABC phenomenon of PEGylated protein drug and even related to the CARPA of COVID-19 vaccine. Therefore, it is urgent to inhibit the generation of APAs and eliminate the ABC phenomenon. Here, "Y-type” PEG was chosen to replace linear PEG due to its weak immunogenicity. "Y-type” PEG-lipid derivatives [DSPE-mPEG2,n (n = 2, 10, and 20 kDa)]-modified doxorubicin liposomes (DOX-PL2,n) and topotecan liposomes (TP-PL2,n) induced lower levels of APAs and could avoid activating complement system. In further research, we found that liposomes decorated with DSPE-mPEG2,n could avoid the ABC phenomenon after duplicate injections. Furthermore, pharmacodynamic tests indicated that DOX-PL2,n and TP-PL2,n improved the curative effect of S180 tumor than DOX-PL2k and TP-PL2k (linear PEGylated liposomes). For the first time, DOX-PL2,n and TP-PL2,n were used for in vivo pharmacokinetic and pharmacodynamic experiments. Liposomes ornamented with "Y-type” PEG may provide new approaches to maintaining long blood circulation time, eliminating the ABC phenomenon of encapsulated active compounds, and also could weaken CARPA and improve tumor therapeutic effect. Our research aims to promote the research and development of "Y-type” PEG-decorated nanocarriers and provide a substantial academic basis for its clinical application.

Inhaled Lipid Nanoparticles Alleviate Established Pulmonary Fibrosis.

Pulmonary fibrosis, a sequela of lung injury resulting from severe infection such as severe acute respiratory syndrome-like coronavirus (SARS-CoV-2) infection, is a kind of life-threatening lung disease with limited therapeutic options. Herein, inhalable liposomes encapsulating metformin, a first-line antidiabetic drug that has been reported to effectively reverse pulmonary fibrosis by modulating multiple metabolic pathways, and nintedanib, a well-known antifibrotic drug that has been widely used in the clinic, are developed for pulmonary fibrosis treatment. The composition of liposomes made of neutral, cationic or anionic lipids, and poly(ethylene glycol) (PEG) is optimized by evaluating their retention in the lung after inhalation. Neutral liposomes with suitable PEG shielding are found to be ideal delivery carriers for metformin and nintedanib with significantly prolonged retention in the lung. Moreover, repeated noninvasive aerosol inhalation delivery of metformin and nintedanib loaded liposomes can effectively diminish the development of fibrosis and improve pulmonary function in bleomycin-induced pulmonary fibrosis by promoting myofibroblast deactivation and apoptosis, inhibiting transforming growth factor 1 (TGFß1) action, suppressing collagen formation, and inducing lipogenic differentiation. Therefore, this work presents a versatile platform with promising clinical translation potential for the noninvasive inhalation delivery of drugs for respiratory disease treatment.

Liposomal Forms of Fluoroquinolones and Antifibrotics Decorated with Mannosylated Chitosan for Inhalation Drug Delivery.

The severe course of COVID-19 leads to the long-terming pulmonary diseases, such as bacterial pneumonia and post-COVID-19 pulmonary fibrosis. Thus, the essential task of biomedicine is a design of new effective drug formulations, including those for inhalation administration. In this work, we propose an approach to the creation of lipid-polymer delivery systems for fluoroquinolones and pirfenidone based on liposomes of various compositions decorated with mucoadhesive mannosylated chitosan. A generalizing study on the physicochemical patterns of the interactions of drugs with bilayers of various compositions was carried out, and the main binding sites were identified. The role of the polymer shell in the stabilization of vesicles and the delayed release of the contents has been demonstrated. For the liquid-polymer formulation of moxifloxacin, a prolonged accumulation of the drug in lung tissues was found after a single endotracheal administration to mice, significantly exceeding the control intravenous and endotracheal administration of the drug.

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.

Approved Nanomedicine against Diseases.

Nanomedicine is a branch of medicine using nanotechnology to prevent and treat diseases. Nanotechnology represents one of the most effective approaches in elevating a drug's treatment efficacy and reducing toxicity by improving drug solubility, altering biodistribution, and controlling the release. The development of nanotechnology and materials has brought a profound revolution to medicine, significantly affecting the treatment of various major diseases such as cancer, injection, and cardiovascular diseases. Nanomedicine has experienced explosive growth in the past few years. Although the clinical transition of nanomedicine is not very satisfactory, traditional drugs still occupy a dominant position in formulation development, but increasingly active drugs have adopted nanoscale forms to limit side effects and improve efficacy. The review summarized the approved nanomedicine, its indications, and the properties of commonly used nanocarriers and nanotechnology.

Use of Microfluidics to Prepare Lipid-Based Nanocarriers.

Lipid-based nanoparticles (LBNPs) are an important tool for the delivery of a diverse set of drug cargoes, including small molecules, oligonucleotides, and proteins and peptides. Despite their development over the past several decades, this technology is still hindered by issues with the manufacturing processes leading to high polydispersity, batch-to-batch and operator-dependent variability, and limits to the production volumes. To overcome these issues, the use of microfluidic techniques in the production of LBNPs has sharply increased over the past two years. Microfluidics overcomes many of the pitfalls seen with conventional production methods, leading to reproducible LBNPs at lower costs and higher yields. In this review, the use of microfluidics in the preparation of various types of LBNPs, including liposomes, lipid nanoparticles, and solid lipid nanoparticles for the delivery of small molecules, oligonucleotides, and peptide/protein drugs is summarized. Various microfluidic parameters, as well as their effects on the physicochemical properties of LBNPs, are also discussed.

Key Design Features of Lipid Nanoparticles and Electrostatic Charge-Based Lipid Nanoparticle Targeting.

Lipid nanoparticles (LNP) have gained much attention after the approval of mRNA COVID-19 vaccines. The considerable number of currently ongoing clinical studies are testament to this fact. These efforts towards the development of LNPs warrant an insight into the fundamental developmental aspects of such systems. In this review, we discuss the key design aspects that confer efficacy to a LNP delivery system, i.e., potency, biodegradability, and immunogenicity. We also cover the underlying considerations regarding the route of administration and targeting of LNPs to hepatic and non-hepatic targets. Furthermore, since LNP efficacy is also a function of drug/nucleic acid release within endosomes, we take a holistic view of charged-based targeting approaches of LNPs not only in the context of endosomal escape but also in relation to other comparable target cell internalization strategies. Electrostatic charge-based interactions have been used in the past as a potential strategy to enhance the drug release from pH-sensitive liposomes. In this review, we cover such strategies around endosomal escape and cell internalization in low pH tumor micro-environments.

Establishment of an Antiplasmodial Vaccine Based on PfRH5-Encoding RNA Replicons Stabilized by Cationic Liposomes.

BACKGROUND: Nucleic acid-based vaccines have been studied for the past four decades, but the approval of the first messenger RNA (mRNA) vaccines during the COVID-19 pandemic opened renewed perspectives for the development of similar vaccines against different infectious diseases. Presently available mRNA vaccines are based on non-replicative mRNA, which contains modified nucleosides encased in lipid vesicles, allowing for entry into the host cell cytoplasm, and reducing inflammatory reactions. An alternative immunization strategy employs self-amplifying mRNA (samRNA) derived from alphaviruses, but lacks viral structural genes. Once incorporated into ionizable lipid shells, these vaccines lead to enhanced gene expression, and lower mRNA doses are required to induce protective immune responses. In the present study, we tested a samRNA vaccine formulation based on the SP6 Venezuelan equine encephalitis (VEE) vector incorporated into cationic liposomes (dimethyldioctadecyl ammonium bromide and a cholesterol derivative). Three vaccines were generated that encoded two reporter genes (GFP and nanoLuc) and the Plasmodium falciparum reticulocyte binding protein homologue 5 (PfRH5). METHODS: Transfection assays were performed using Vero and HEK293T cells, and the mice were immunized via the intradermal route using a tattooing device. RESULTS: The liposome-replicon complexes showed high transfection efficiencies with in vitro cultured cells, whereas tattooing immunization with GFP-encoding replicons demonstrated gene expression in mouse skin up to 48 h after immunization. Mice immunized with liposomal PfRH5-encoding RNA replicons elicited antibodies that recognized the native protein expressed in P. falciparum schizont extracts, and inhibited the growth of the parasite in vitro. CONCLUSION: Intradermal delivery of cationic lipid-encapsulated samRNA constructs is a feasible approach for developing future malaria vaccines.

Neutralization of the new coronavirus by extracting their spikes using engineered liposomes.

The devastating COVID-19 pandemic motivates the development of safe and effective antivirals to reduce morbidity and mortality associated with infection. We developed nanoscale liposomes that are coated with the cell receptor of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus that causes COVID-19. Lentiviral particles pseudotyped with the spike protein of SARS-CoV-2 were constructed and used to test the virus neutralization potential of the engineered liposomes. Under TEM, we observed for the first time a dissociation of spike proteins from the pseudovirus surface when the pseudovirus was purified. The liposomes potently inhibit viral entry into host cells by extracting the spike proteins from the pseudovirus surface. As the receptor on the liposome surface can be readily changed to target other viruses, the receptor-coated liposome represents a promising strategy for broad spectrum antiviral development.

Microfluidics for nano-drug delivery systems: from fundamentals to industrialization

In recent years, owing to the miniaturization of the fluidic environment, microfluidic technology offers unique opportunities for the implementation of nano drug delivery systems (NDDSs) production processes. Compared with traditional methods, microfluidics improves the controllability and uniformity of NDDSs. The fast mixing and laminar flow properties achieved in the microchannels can tune the physicochemical properties of NDDSs, including particle size, distribution and morphology, resulting in narrow particle size distribution and high drug-loading capacity. The success of lipid nanoparticles encapsulated mRNA vaccines against coronavirus disease 2019 by microfluidics also confirmed its feasibility for scaling up the preparation of NDDSs via parallelization or numbering-up. In this review, we provide a comprehensive summary of microfluidics-based NDDSs, including the fundamentals of microfluidics, microfluidic synthesis of NDDSs, and their industrialization. The challenges of microfluidics-based NDDSs in the current status and the prospects for future development are also discussed. We believe that this review will provide good guidance for microfluidics-based NDDSs.Copyright © 2023 Chinese Pharmaceutical Association and Institute of Materia Medica, Chinese Academy of Medical Sciences

Proof-of-Concept Study of Liposomes with a Set of SARS-CoV-2 Viral Peptidic T-Cell Epitopes as a Vaccine

Abstract: Potential nonameric epitopes of CD8+ T lymphocytes were selected from the composition of structural, accessory, and nonstructural proteins of the SARS-CoV-2 virus (13 peptides) and a 15-mer epitope of CD4+ T lymphocytes, from the S-protein, based on the analysis of publications on genome-wide immunoinformatic analysis of T-cell epitopes of the virus (Wuhan strain), as well as a number of clinical studies of immunodominant epitopes among patients recovering from COVID-19 disease. The peptides were synthesized and five compositions of 6–7 peptides were included in liposomes from egg phosphatidylcholine and cholesterol (~200 nm size) obtained by extrusion. After double subcutaneous immunization of conventional mice, activation of cellular immunity was assessed by the level of cytokine synthesis by splenocytes in vitro in response to stimulation with relevant peptide compositions. Liposomal formulation exhibiting the best result in terms of the formation of specific cellular immunity in response to vaccination was selected for further experiments. Evaluation of the protective efficacy of this formulation in an infectious mouse model showed a positive trend in the frequency of occurrence of hyaline-like membranes in the lumen of the alveoli, as well as a somewhat lower severity of microcirculatory disorders. The latter circumstance can potentially help reduce the severity of the disease and prevent its adverse outcomes. A method to produce liposome preparations with peptide compositions for long-term storage is under development. © 2022, Pleiades Publishing, Ltd.

Recent Developments in Oral Delivery of Vaccines Using Nanocarriers.

As oral administration of vaccines is the preferred route due to its high patient compliance and ability to stimulate both cellular and humoral immune responses, it is also associated with several challenges that include denaturation of vaccine components in the acidic environment of the stomach, degradation from proteolytic enzymes, and poor absorption through the intestinal membrane. To achieve effective delivery of such biomolecules, there is a need to investigate novel strategies of formulation development that can overcome the barriers associated with conventional vaccine delivery systems. Nanoparticles are advanced drug delivery carriers that provide target-oriented delivery by encapsulating vaccine components within them, thus making them stable against unfavorable conditions. This review provides a detailed overview of the different types of nanocarriers and various approaches that can enhance oral vaccine delivery.

Delivery of anti-microRNA-21 by lung-targeted liposomes for pulmonary fibrosis treatment.

Idiopathic pulmonary fibrosis (IPF) is a chronic lung disorder with a low survival rate. Pulmonary fibrosis is one of the complications of COVID-19 and has a high prevalence in COVID-19 patients. Currently, no effective therapies other than lung transplantation are available to cure IPF and post-COVID-19 pulmonary fibrosis. MicroRNAs are small non-coding RNAs that mediate the development and progression of pulmonary fibrosis, thus making them potent drug candidates for this serious disease. MicroRNA-21 (miR-21) promotes not only the differentiation of fibroblasts to myofibroblasts but also epithelial-mesenchymal transition, both of which have been proposed as fundamental processes in pulmonary fibrosis development. Delivery of anti-miR-21 to block the miR-21-associated fibrogenic pathways represents a promising therapy for pulmonary fibrosis. However, microRNA treatment is challenged by quick degradation of RNA in blood, poor cellular uptake, and off-target effects. To overcome these challenges, we developed a lung-targeted, cationic liposome formulation to encapsulate anti-miR-21, enhance its delivery efficiency, and improve the therapeutic efficacy. We optimized the liposome formulation and demonstrated the anti-fibrotic effects using both in vitro and in vivo lung fibrosis models. Our results showed that anti-miR-21 delivered by cationic liposomes suppressed myofibroblast differentiation, reduced the synthesis of extracellular matrix, and inhibited fibrosis progression.

Self-driving laboratories: A paradigm shift in nanomedicine development.

Nanomedicines have transformed promising therapeutic agents into clinically approved medicines with optimal safety and efficacy profiles. This is exemplified by the mRNA vaccines against COVID-19, which were made possible by lipid nanoparticle technology. Despite the success of nanomedicines to date, their design remains far from trivial, in part due to the complexity associated with their preclinical development. Herein, we propose a nanomedicine materials acceleration platform (NanoMAP) to streamline the preclinical development of these formulations. NanoMAP combines high-throughput experimentation with state-of-the-art advances in artificial intelligence (including active learning and few-shot learning) as well as a web-based application for data sharing. The deployment of NanoMAP requires interdisciplinary collaboration between leading figures in drug delivery and artificial intelligence to enable this data-driven design approach. The proposed approach will not only expedite the development of next-generation nanomedicines but also encourage participation of the pharmaceutical science community in a large data curation initiative.

Emerging Trends in Lipid-Based Vaccine Delivery: A Special Focus on Developmental Strategies, Fabrication Methods, and Applications.

Lipid-based vaccine delivery systems such as the conventional liposomes, virosomes, bilosomes, vesosomes, pH-fusogenic liposomes, transferosomes, immuno-liposomes, ethosomes, and lipid nanoparticles have gained a remarkable interest in vaccine delivery due to their ability to render antigens in vesicular structures, that in turn prevents its enzymatic degradation in vivo. The particulate form of lipid-based nanocarriers confers immunostimulatory potential, making them ideal antigen carriers. Facilitation in the uptake of antigen-loaded nanocarriers, by the antigen-presenting cells and its subsequent presentation through the major histocompatibility complex molecules, leads to the activation of a cascade of immune responses. Further, such nanocarriers can be tailored to achieve the desired characteristics such as charge, size, size distribution, entrapment, and site-specificity through modifications in the composition of lipids and the selection of the appropriate method of preparation. This ultimately adds to its versatility as an effective vaccine delivery carrier. The current review focuses on the various lipid-based carriers that have been investigated to date as potential vaccine delivery systems, the factors that affect their efficacy, and their various methods of preparation. The emerging trends in lipid-based mRNA vaccines and lipid-based DNA vaccines have also been summarized.

Similarities and differences of chemical compositions and physical and functional properties of adjuvant system 01 and army liposome formulation with QS21.

A vaccine adjuvant known as Adjuvant System 01 (AS01) consists of liposomes containing a mixture of natural congeners of monophosphoryl lipid A (MPL®) obtained from bacterial lipopolysaccharide, and a tree saponin known as QS21. Two vaccines containing AS01 as the adjuvant have been licensed, including a malaria vaccine (Mosquirix®) approved by World Health. Organization and European Medicines Agency for use in sub-Saharan Africa, and a shingles vaccine (Shingrix®) approved by the U.S. Food and Drug Administration. The success of the AS01 vaccine adjuvant has led to the development of another liposomal vaccine adjuvant, referred to as Army Liposome Formulation with QS21 (ALFQ). Like AS01, ALFQ consists of liposomes containing monophosphoryl lipid A (as a synthetic molecule known as 3D-PHAD®) and QS21 as adjuvant constituents, and the polar headgroups of the liposomes of AS01 and ALFQ are similar. We compare here AS01 with ALFQ with respect to their similar and different liposomal chemical structures and physical characteristics with a goal of projecting some of the likely mechanisms of safety, side effects, and mechanisms of adjuvanticity. We hypothesize that some of the side effects exhibited in humans after injection of liposome-based vaccines might be caused by free fatty acid and lysophospholipid released by enzymatic attack of liposomal phospholipid by phospholipase A2 at the injection site or systemically after injection.

One-Step Formation of Targeted Liposomes in a Versatile Microfluidic Mixing Device.

Targeted liposomes, as a promising carrier, have received tremendous attention in COVID-19 vaccines, molecular imaging, and cancer treatment, due to their enhanced cellular uptake and payload accumulation at target sites. However, the conventional methods for preparing targeted liposomes still suffer from limitations, including complex operation, time-consuming, and poor reproducibility. Herein, a facile and scalable strategy is developed for one-step construction of targeted liposomes using a versatile microfluidic mixing device (MMD). The engineered MMD provides an advanced synthesis platform for multifunctional liposome with high production rate and controllability. To validate the method, a programmed death-ligand 1 (PD-L1)-targeting aptamer modified indocyanine green (ICG)-liposome (Apt-ICG@Lip) is successfully constructed via the MMD. ICG and the PD-L1-targeting aptamer are used as model drug and targeting moiety, respectively. The Apt-ICG@Lip has high encapsulation efficiency (89.9 ± 1.4%) and small mean diameter (129.16 ± 5.48 nm). In vivo studies (PD-L1-expressing tumor models) show that Apt-ICG@Lip can realize PD-L1 targeted photoacoustic imaging, fluorescence imaging, and photothermal therapy. To verify the versatility of this approach, various targeted liposomes with different functions are further prepared and investigated. These experimental results demonstrate that this method is concise, efficient, and scalable to prepare multifunctional targeted liposomal nanoplatforms for molecular imaging and disease theranostics.

Liposomes and liposome-like nanoparticles: From anti-fungal infection to the COVID-19 pandemic treatment.

The liposome is the first nanomedicine transformed into the market and applied to human patients. Since then, such phospholipid bilayer vesicles have undergone technological advancements in delivering small molecular-weight compounds and biological drugs. Numerous investigations about liposome uses were conducted in different treatment fields, including anti-tumor, anti-fungal, anti-bacterial, and clinical analgesia, owing to liposome's ability to reduce drug cytotoxicity and improve the therapeutic efficacy and combinatorial delivery. In particular, two liposomal vaccines were approved in 2021 to combat COVID-19. Herein, the clinically used liposomes are reviewed by introducing various liposomal preparations in detail that are currently proceeding in the clinic or on the market. Finally, we will discuss the challenges of developing liposomes and cutting-edge liposomal delivery for biological drugs and combination therapy.

Potent Therapeutic Strategies for COVID-19 with Single-Domain Antibody Immunoliposomes Neutralizing SARS-CoV-2 and Lip/cGAMP Enhancing Protective Immunity.

The worldwide spread of COVID-19 continues to impact our lives and has led to unprecedented damage to global health and the economy. This highlights the need for an efficient approach to rapidly develop therapeutics and prophylactics against SARS-CoV-2. We modified a single-domain antibody, SARS-CoV-2 VHH, to the surface of the liposomes. These immunoliposomes demonstrated a good neutralizing ability, but could also carry therapeutic compounds. Furthermore, we used the 2019-nCoV RBD-SD1 protein as an antigen with Lip/cGAMP as the adjuvant to immunize mice. Lip/cGAMP enhanced the immunity well. It was demonstrated that the combination of RBD-SD1 and Lip/cGAMP was an effective preventive vaccine. This work presented potent therapeutic anti-SARS-CoV-2 drugs and an effective vaccine to prevent the spread of COVID-19.

From Olive Oil Emulsions to COVID-19 Vaccines: Liposomes Came First.

It has been a long journey from Pliny the Elder (23-79 AD) to the FDA approval of the first injectable nanomedicine in 1997. A journey powered by intellectual curiosity, which began with sprinkling olive oil on seawater and culminated in playing around with smears of egg lecithin on microscopic slides. This brief review highlights how a few pairs of gifted hands attached to highly motivated brains have turned a curious discovery made under a microscopic lens into novel nanotherapeutics including liposome-based anti-cancer drugs and potent liposomal vaccines given to millions.