Vaccine-Induced Immunity Elicited by Microneedle Delivery of Influenza Ectodomain Matrix Protein 2 Virus-like Particle (M2e VLP)-Loaded PLGA Nanoparticles.

This study focused on developing an influenza vaccine delivered in polymeric nanoparticles (NPs) using dissolving microneedles. We first formulated an influenza extracellular matrix protein 2 virus-like particle (M2e VLP)-loaded with poly(lactic-co-glycolic) acid (PLGA) nanoparticles, yielding M2e5x VLP PLGA NPs. The vaccine particles were characterized for their physical properties and in vitro immunogenicity. Next, the M2e5x VLP PLGA NPs, along with the adjuvant Alhydrogel® and monophosphoryl lipid A® (MPL-A®) PLGA NPs, were loaded into fast-dissolving microneedles. The vaccine microneedle patches were then evaluated in vivo in a murine model. The results from this study demonstrated that the vaccine nanoparticles effectively stimulated antigen-presenting cells in vitro resulting in enhanced autophagy, nitric oxide, and antigen presentation. In mice, the vaccine elicited M2e-specific antibodies in both serum and lung supernatants (post-challenge) and induced significant expression of CD4+ and CD8+ populations in the lymph nodes and spleens of immunized mice. Hence, this study demonstrated that polymeric particulates for antigen and adjuvant encapsulation, delivered using fast-dissolving microneedles, significantly enhanced the immunogenicity of a conserved influenza antigen.

Laser-assisted intradermal delivery of a microparticle vaccine for respiratory syncytial virus induces a robust immune response.

Respiratory syncytial virus (RSV) is an infectious disease that poses a significant public health risk in young children. Vaccine studies conducted in the 1960s using an intramuscular injection of formalin-inactivated respiratory syncytial virus (Fi-RSV) resulted in an enhanced respiratory disease and led to the failure of the vaccine. Thus, the virus-like particles (VLP) of the RSV fusion (F) protein was used as the vaccine antigen in this study. The F-VLP was encapsulated in a microparticle (MP) matrix composed of cross-linked bovine serum albumin (BSA) to enhance the antigen presentation and uptake. Moreover, a painless vaccination method would be desirable for an infectious disease that mainly affects young children. Thus, an ablative laser device, Precise Laser Epidermal System (P.L.E.A.S.E), was utilized to create micropores on the skin for vaccine delivery. We observed enhanced antigen presentation of the vaccine microparticles (F-VLP MP) with and without the adjuvant monophosphoryl lipid A (MPL-A) MP in dendritic cells. Consequently, Swiss Webster mice were immunized with the adjuvanted vaccine microparticles using the P.L.E.A.S.E laser to study the in vivo immunogenicity. The immunized mice had high serum immunoglobulin (IgG, IgG2a) levels, indicating a Th1 response. Subsequent analysis of lung homogenates post- RSV challenge revealed high IgA, indicating generation of a mucosal immune response upon intradermal immunization. Flowcytometry analysis showed high CD8+, and CD4+ expression in the lymph node and spleen of the adjuvanted vaccine microparticle immunized mice. Increased expression of interferon gamma (IFN-γ) in the spleen cells further proved Th1 polarized immune response. Finally, an immune plaque assay indicated significantly low lung viral titer in the mice immunized with intradermal adjuvanted vaccine microparticles. Thus, ablative laser-assisted immunization with the F-VLP based adjuvanted vaccine microparticles could be a promising vaccine candidate for RSV.

An Overview of Current Accomplishments and Gaps of COVID-19 Vaccine Platforms and Considerations for Next Generation Vaccines.

Vaccines against SARS-CoV-2 have transformed the course of the COVID-19 pandemic with more than 30 authorizations. More than 2 billion people have been vaccinated with these vaccines developed on very different manufacturing platforms. We have reviewed the unprecedented work done in various aspects of the authorized vaccines and listed three potential improvements: 1) long-term stability at room-temperature conditions; 2) suitability for diverse populations such as infants, elderly, immune-compromised, and those with pre-existing or ongoing diseases; and 3) ability to act against different strains. In this article, we have discussed the current status of COVID-19 vaccines with respect to 1) diversity (strength and breadth) of initial immune responses and long-term immune memory; 2) prime-boost combinations that induce protection against variants; and 3) breakthrough infections. Further, we have listed host, product (critical quality attributes), and viral pathogenic factors that contribute to safety, efficacy, and effectiveness of vaccines. In addition, we have elaborated on the potential to (develop models and) determine the immune correlates that can predict long-term immune memory. The graphical representation of the abstract is provided as Fig. 1.

Adjuvanted-SARS-CoV-2 Spike Protein-Based Microparticulate Vaccine Delivered by Dissolving Microneedles Induces Humoral, Mucosal, and Cellular Immune Responses in Mice.

COVID-19 continues to cause an increase in the number of cases and deaths worldwide. Due to the ever-mutating nature of the virus, frequent vaccination against COVID-19 is anticipated. Most of the approved SARS-CoV-2 vaccines are administered using the conventional intramuscular route, causing vaccine hesitancy. Thus, there is a need for an effective, non-invasive vaccination strategy against COVID-19. This study evaluated the synergistic effects of a subunit microparticulate vaccine delivered using microneedles. The microparticles encapsulated a highly immunogenic subunit protein of the SARS-CoV-2 virus, such as the spike protein's receptor binding domain (RBD). Adjuvants were also incorporated to enhance the spike RBD-specific immune response. Our vaccination study reveals that a microneedle-based vaccine delivering these microparticles induced spike RBD-specific IgM, IgG, IgG1, IgG2a, and IgA antibodies. The vaccine also generated high levels of CD4+ and CD8a+ molecules in the secondary lymphoid organs. Overall, dissolving microneedles delivery spike RBD antigen in microparticulate form induced a robust immune response, paving the way for an alternative self-administrable, non-invasive vaccination strategy against COVID-19.

Subunit microparticulate vaccine delivery using microneedles trigger significant SARS-spike-specific humoral and cellular responses in a preclinical murine model.

The objective of this "proof-of-concept" study was to evaluate the synergistic effect of a subunit microparticulate vaccine and microneedles (MN) assisted vaccine delivery system against a human coronavirus. Here, we formulated PLGA polymeric microparticles (MPs) encapsulating spike glycoprotein (GP) of SARS-CoV as the model antigen. Similarly, we formulated adjuvant MPs encapsulating Alhydrogel® and AddaVax™. The antigen/adjuvant MPs were characterized and tested in vitro for immunogenicity. We found that the antigen/adjuvant MPs were non-cytotoxic in vitro. The spike GP MPs + Alhydrogel® MPs + AddaVax™ MPs showed enhanced immunogenicity in vitro as confirmed through the release of nitrite, autophagy, and antigen presenting molecules with their co-stimulatory molecules. Next, we tested the in vivo efficacy of the spike GP MP vaccine with and without adjuvant MPs in mice vaccinated using MN. The spike GP MPs + Alhydrogel® MPs + AddaVax™ MPs induced heightened spike GP-specific IgG, IgG1 and IgG2a antibodies in mice. Also, spike GP MPs + Alhydrogel® MPs + AddaVax™ MPs enhanced expression of CD4+ and CD8+ T cells in secondary lymphoid organ like spleen. These results indicated spike GP-specific humoral immunity and cellular immunity in vivo. Thus, we employed the benefits of both the subunit vaccine MPs and dissolving MN to form a non-invasive and effective vaccination strategy against human coronaviruses.

Dissolving Microneedles Loaded with Nanoparticle Formulation of Respiratory Syncytial Virus Fusion Protein Virus-like Particles (F-VLPs) Elicits Cellular and Humoral Immune Responses.

Respiratory syncytial virus (RSV) is one of the leading causes of bronchiolitis and pneumonia in children ages five years and below. Recent outbreaks of the virus have proven that RSV remains a severe burden on healthcare services. Thus, a vaccine for RSV is a need of the hour. Research on novel vaccine delivery systems for infectious diseases such as RSV can pave the road to more vaccine candidates. Among many novel vaccine delivery systems, a combined system with polymeric nanoparticles loaded in dissolving microneedles holds a lot of potential. In this study, the virus-like particles of the RSV fusion protein (F-VLP) were encapsulated in poly (D, L-lactide-co-glycolide) (PLGA) nanoparticles (NPs). These NPs were then loaded into dissolving microneedles (MNs) composed of hyaluronic acid and trehalose. To test the in vivo immunogenicity of the nanoparticle-loaded microneedles, Swiss Webster mice were immunized with the F-VLP NPs, both with and without adjuvant monophosphoryl lipid A (MPL) NPs loaded in the MN. The mice immunized with the F-VLP NP + MPL NP MN showed high immunoglobulin (IgG and IgG2a) levels both in the serum and lung homogenates. A subsequent analysis of lung homogenates post-RSV challenge revealed high IgA, indicating the generation of a mucosal immune response upon intradermal immunization. A flowcytometry analysis showed high CD8+ and CD4+ expression in the lymph nodes and spleens of the F-VLP NP + MPL NP MN-immunized mice. Thus, our vaccine elicited a robust humoral and cellular immune response in vivo. Therefore, PLGA nanoparticles loaded in dissolving microneedles could be a suitable novel delivery system for RSV vaccines.

Gonococcal microparticle vaccine in dissolving microneedles induced immunity and enhanced bacterial clearance in infected mice.

There is an alarming rise in the number of gonorrhea cases worldwide. Neisseria gonorrhoeae, the bacteria that causes gonorrhea infection, has gradually developed antimicrobial resistance over the years. To date, there is no licensed vaccine for gonorrhea. This study investigates the in vivo immunogenicity of a whole-cell inactivated gonococci in a microparticle formulation (Gc-MP) along with adjuvant microparticles (Alhydrogel®- Alum MP and AddaVax™ MP) delivered transdermally using dissolving microneedles (MN). The proposed vaccine formulation (Gc-MP + Alum MP + AddaVax™ MP) was assessed for induction of humoral, cellular, and protective immune responses in vivo. Our results show the induction of significant gonococcal-specific serum IgG, IgG1, IgG2a, and vaginal mucosal IgA antibodies in mice immunized with Gc-MP + Alum MP + AddaVax™ MP and Gc-MP when compared to the control groups receiving blank MN or no treatment. The serum bactericidal assay revealed that the antibodies generated in mice after immunization with Gc-MP + Alum MP + AddaVax™ MP were bactericidal towards live Neisseria gonorrhoeae. Gc-MP + Alum MP + AddaVax™ MP and Gc-MP-immunized mice showed enhanced clearance rate of gonococcal bacterial infection post challenge. In contrast, the control groups did not begin to clear the infection until day 10. In addition, the mice which received Gc-MP + Alum MP + AddaVax™ MP showed enhanced expression of cellular immunity markers CD4 and CD8 on the surface of T cells in the spleen and lymph nodes. Taken together, the data shows that microneedle immunization with whole-cell inactivated gonococci MP in mice induced humoral, cellular, and protective immunity against gonococcal infection.

An Adjuvanted Inactivated SARS-CoV-2 Microparticulate Vaccine Delivered Using Microneedles Induces a Robust Immune Response in Vaccinated Mice.

SARS-CoV-2, the causal agent of COVID-19, is a contagious respiratory virus that frequently mutates, giving rise to variant strains and leading to reduced vaccine efficacy against the variants. Frequent vaccination against the emerging variants may be necessary; thus, an efficient vaccination system is needed. A microneedle (MN) vaccine delivery system is non-invasive, patient-friendly, and can be self-administered. Here, we tested the immune response produced by an adjuvanted inactivated SARS-CoV-2 microparticulate vaccine administered via the transdermal route using a dissolving MN. The inactivated SARS-CoV-2 vaccine antigen and adjuvants (Alhydrogel® and AddaVax™) were encapsulated in poly(lactic-co-glycolic acid) (PLGA) polymer matrices. The resulting MP were approximately 910 nm in size, with a high percentage yield and percent encapsulation efficiency of 90.4%. In vitro, the vaccine MP was non-cytotoxic and increased the immunostimulatory activity measured as nitric oxide release from dendritic cells. The adjuvant MP potentiated the immune response of the vaccine MP in vitro. In vivo, the adjuvanted SARS-CoV-2 MP vaccine induced high levels of IgM, IgG, IgA, IgG1, and IgG2a antibodies and CD4+ and CD8+ T-cell responses in immunized mice. In conclusion, the adjuvanted inactivated SARS-CoV-2 MP vaccine delivered using MN induced a robust immune response in vaccinated mice.

Zika Vaccine Microparticles (MPs)-Loaded Dissolving Microneedles (MNs) Elicit a Significant Immune Response in a Pre-Clinical Murine Model.

Although the global Zika epidemic in 2015-16 fueled vaccine development efforts, there is no approved Zika vaccine or treatment available to date. Current vaccine platforms in clinical trials are administered via either subcutaneous or intramuscular injections, which are painful and decrease compliance. Therefore, in the present study, we explored Zika vaccine microparticles (MPs)-loaded dissolving microneedles (MNs) with adjuvant MPs encapsulating Alhydrogel® and MPL-A® administered via the transdermal route as a pain-free vaccine strategy. We characterized the MNs for needle length, pore formation, and dissolvability when applied to murine skin. Further, we evaluated the in vivo efficacy of vaccine MPs-loaded MNs with or without adjuvants by measuring the immune response after transdermal immunization. The vaccine MPs-loaded dissolving MNs with adjuvants induced significant IgG, IgG1, and IgG2a titers in immunized mice compared to the untreated control group. After the dosing regimen, the animals were challenged with Zika virus, monitored for seven days, and sacrificed to collect spleen and lymph nodes. The lymphocytes and splenocytes from the immunized mice showed significant expressions of helper (CD4) and cytotoxic (CD8a) cell surface markers compared to the control group. Thus, this study puts forth a 'proof-of-concept' for a pain-free transdermal vaccine strategy against Zika.

Nanoparticle formulation of the fusion protein virus like particles of respiratory syncytial virus stimulates enhanced in vitro antigen presentation and autophagy.

Respiratory Syncytial Virus (RSV) is one of the leading causes of bronchiolitis and pneumonia in childrenunder one year globally. As a result, RSV poses a severe burden on healthcare services. Thus, a vaccine for RSV is a global need. Utilizing polymeric nanoparticles as a delivery system for vaccine antigen holds a lot of promise. In this study, the virus like particles of RSV fusion protein (F-VLP) was encapsulated in poly (D, L-lactide-co-glycolide) (PLGA) nanoparticles (NP). The F-VLP NP was formulated using a double emulsion solvent evaporation technique. The optimized NPs had a particle size of 525 ± 10.5 nm and an antigen encapsulation efficiency of 73% ± 10.5. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the F-VLP was stable post formulation. The F-VLP NP showed a sustained release of the F-VLP antigen for up to a week. In vitro study revealed that the F-VLP NP were non-cytotoxic, and the cellular uptake of the NPs by dendritic cells was observed within 3 h. The F-VLP NP with adjuvant monophosphoryl lipid A (MPL) NP and without MPL NP showed enhanced expression of antigen presentation molecule major histocompatibility complex (MHC)-I and autophagosomes in dendritic cells. In summary, the sustained release of the antigen from the F-VLP NP and the particulate nature of the vaccine resulted in enhanced antigen presentation and induction of autophagy in antigen-presenting cells (APCs).

A dual-delivery platform for vaccination using antigen-loaded nanoparticles in dissolving microneedles.

Effective vaccines delivered via painless methods would revolutionize the way people approach vaccinations. This study focused on the development of fast-dissolving microneedles (MNs) to deliver antigen-loaded sustained release polymeric nanoparticles (NPs), achieving a dual-delivery platform for vaccination through the skin. The platform utilizes dissolving MNs (dMNs), which penetrate to the epidermal layer of the skin and rapidly dissolve, releasing the antigen-loaded NPs. In this study, seven dissolving microneedle formulations were tested based on screening of various biocompatible and biodegradable polymers and sugars. The lead dMN formulation was selected based on optimal mechanical strength and dissolution of the needles and was loaded with poly(lactic-co-glycolic) acid (PLGA) NPs encapsulating a model influenza matrix 2 (M2) protein antigen. Antigen-loading efficiency in the needles was determined by centrifugation of the lead formulation containing various concentrations of antigen nanoparticles. Next, the reproducibility and translatability of ex vivo mechanical strength and dissolvability of the lead M2 PLGA NP-loaded dMN formulation was assessed by formulating and testing two different microneedle arrays on murine and porcine skin. Finally, the lead microneedle array was loaded with fluorescent dye NPs and evaluated for pore formation and closure in vivo in a murine model. This proof-of-concept study yielded an easy-to-formulate, well-characterized, translatable antigen NP-loaded dMN platform for transdermal vaccine administration.

Enhanced Immunogenicity of an Influenza Ectodomain Matrix-2 Protein Virus-like Particle (M2e VLP) Using Polymeric Microparticles for Vaccine Delivery.

In this study, we demonstrate how encapsulating a conserved influenza ectodomain matrix-2 protein virus-like particle (M2e5x VLP) into a pre-crosslinked bovine serum albumin (BSA) polymeric matrix enhances in vitro antigen immunogenicity and in vivo efficacy. The spray-dried M2e5x VLP-loaded BSA microparticles (MPs) showed enhanced stimulation of antigen presenting cells (APCs), as confirmed through nitrite production and increased antigen-cell interactions seen in real time using live-cell imaging. Next, to further boost the immunogenicity of M2e5x VLP microparticles, M2e5x MPs were combined with Alhydrogel® and monophosphoryl lipid-A (MPL-A®) adjuvant microparticles. M2e5x VLP MPs and the combination VLP M2e5x VLP + Alhydrogel® + MPL-A® MPs elicited a significant increase in the expression of antigen-presenting molecules in dendritic cells compared to M2e5x VLP alone. Lastly, for preliminary evaluation of in vivo efficacy, the vaccine was administered in mice through the skin using an ablative laser. The M2e5x VLP + Alhydrogel® + MPL-A® MPs were shown to induce high levels of M2e-specific IgG antibodies. Further, a challenge with live influenza revealed heightened T-cell stimulation in immune organs of mice immunized with M2e5x VLP + Alhydrogel® + MPL-A® MPs. Hence, we utilized the advantages of both VLP and polymeric delivery platforms to enhance antigen immunogenicity and adaptive immunity in vivo.

Novel microparticulate Zika vaccine induces a significant immune response in a preclinical murine model after intramuscular administration.

Despite the detrimental effects associated with Zika infection, there are no approved treatments or vaccines available. To address the need for a safe and effective vaccine for Zika, we formulated poly(lactic-co-glycolic) acid (PLGA) polymeric vaccine microparticles (MP) encapsulating the inactivated Zika virus, along with adjuvant MP encapsulating Alhydrogel® and MPL-A®. We characterized the vaccine MP for size, surface charge, morphology, encapsulation efficiency, and antigen integrity. Further, we evaluated immunogenicity and cytotoxicity of vaccine MP in vitro in murine dendritic cells. Vaccine MP with adjuvants induced significantly higher production of nitric oxide, a marker of innate immunity, when compared to the untreated cells. In addition, vaccine MP with or without adjuvants induced increased autophagy in murine dendritic cells when compared to inactivated Zika virus, which is critical in antigen presentation. Next, we evaluated in vivo efficacy of vaccine MP with and without adjuvants in a preclinical murine model by measuring the immune response after intramuscular administration. Vaccine MP with adjuvants induced significant IgG, Ig2a, and IgG1 titers as compared to the control group of untreated mice. Thus, this study provided the 'proof-of-concept' for a microparticulate Zika vaccine.

Microneedle Delivery of an Adjuvanted Microparticulate Vaccine Induces High Antibody Levels in Mice Vaccinated against Coronavirus.

This 'proof-of-concept' study aimed to test the microparticulate vaccine delivery system and a transdermal vaccine administration strategy using dissolving microneedles (MN). For this purpose, we formulated poly(lactic-co-glycolic) acid (PLGA) microparticles (MP) encapsulating the inactivated canine coronavirus (iCCoV), as a model antigen, along with adjuvant MP encapsulating Alhydrogel® and AddaVax. We characterized the vaccine MP for size, surface charge, morphology, and encapsulation efficiency. Further, we evaluated the in vitro immunogenicity, cytotoxicity, and antigen-presentation of vaccine/adjuvant MP in murine dendritic cells (DCs). Additionally, we tested the in vivo immunogenicity of the MP vaccine in mice through MN administration. We evaluated the serum IgG, IgA, IgG1, and IgG2a responses using an enzyme-linked immunosorbent assay. The results indicate that the particulate form of the vaccine is more immunogenic than the antigen suspension in vitro. We found the vaccine/adjuvant MP to be non-cytotoxic to DCs. The expression of antigen-presenting molecules, MHC I/II, and their costimulatory molecules, CD80/40, increased with the addition of the adjuvants. Moreover, the results suggest that the MP vaccine is cross presented by the DCs. In vivo, the adjuvanted MP vaccine induced increased antibody levels in mice following vaccination and will further be assessed for its cell-mediated responses.

Microneedles: A New Generation Vaccine Delivery System.

Transdermal vaccination route using biodegradable microneedles is a rapidly progressing field of research and applications. The fear of painful needles is one of the primary reasons most people avoid getting vaccinated. Therefore, developing an alternative pain-free method of vaccination using microneedles has been a significant research area. Microneedles comprise arrays of micron-sized needles that offer a pain-free method of delivering actives across the skin. Apart from being pain-free, microneedles provide various advantages over conventional vaccination routes such as intramuscular and subcutaneous. Microneedle vaccines induce a robust immune response as the needles ranging from 50 to 900 µm in length can efficiently deliver the vaccine to the epidermis and the dermis region, which contains many Langerhans and dendritic cells. The microneedle array looks like band-aid patches and offers the advantages of avoiding cold-chain storage and self-administration flexibility. The slow release of vaccine antigens is an important advantage of using microneedles. The vaccine antigens in the microneedles can be in solution or suspension form, encapsulated in nano or microparticles, and nucleic acid-based. The use of microneedles to deliver particle-based vaccines is gaining importance because of the combined advantages of particulate vaccine and pain-free immunization. The future of microneedle-based vaccines looks promising however, addressing some limitations such as dosing inadequacy, stability and sterility will lead to successful use of microneedles for vaccine delivery. This review illustrates the recent research in the field of microneedle-based vaccination.

Laser-assisted skin delivery of immunocontraceptive rabies nanoparticulate vaccine in poloxamer gel.

A painless skin delivery of vaccine for disease prevention is of great advantage in improving compliance in patients. To test this idea as a proof of concept, we utilized a pDNA vaccine construct, pDNAg333-2GnRH that has a dual function of controlling rabies and inducing immunocontraception in animals. The pDNA was administered to mice in a nanoparticulate form delivered through the skin using the P.L.E.A.S.E.® (Precise Laser Epidermal System) microporation laser device. Laser application was well tolerated, and mild skin reaction was healed completely in 8 days. We demonstrated that adjuvanted nanoparticulate pDNA vaccine significantly upregulated the expression of co-stimulatory molecules in dendritic cells. After topical administration of the adjuvanted nano-vaccine in mice, the high avidity serum for GnRH antibodies were induced and maintained up to 9 weeks. The induced immune response was of a mixed Th1/Th2 profile as measured by IgG subclasses (IgG2a and IgG1) and cytokine levels (IFN-γ and IL-4). Using flow cytometry, we revealed an increase of CD8+ T-cells and CD45R B cells upon the administration of the adjuvanted vaccine. Our previous study used the same pDNA nanoparticulate vaccine through an IM route, and a comparable immune response was induced using P.L.E.A.S.E. However, the vaccine dose in the current study was four-fold less than what was applied through the IM route.We concluded that laser-assisted skin vaccination has a potential of becoming a safe and reliable vaccination tool for rabies vaccination in animals or even in humans for pre- or post-exposure prophylaxis.

PEGylated Liposomes of Meloxicam: Optimization by Quality by Design, in vitro Characterization and Cytotoxicity Evaluation.

BACKGROUND: Chemoprevention is a strategy which uses drugs which are traditionally not used as anti-cancer drugs; however, they prevent the carcinogenesis. Meloxicam (MLX) is traditionally used as a non-steroidal anti-inflammatory drug (NSAID), but it has been proven to have activity against colorectal cancer. Subsequently MLX seems to be a likely candidate to be utilized in the chemopreventive therapy of colorectal cancer. However, MLX poses shortcomings with respect to its dose required to elicit cytotoxicity. To improve the formulation, we used Quality by design (QbD) for optimization. QbD is a method that employs quality-improving scientific methods that build quality into the formulation by isolating the factors which affect the critical quality attributes of the formulation. The aim of the present study was to utilize the principles of QbD to formulate MLX into a formulation so as to exploit its potential to the fullest. METHODS: Conventional (CLM) and PEGylated liposomes (MPL) of MLX was prepared using hydrogenated soya phosphatidylcholine (HSPC), distearyl phosphatidyl glycerol (DSPG), cholesterol and 1, 2-distearoyl- phosphatidylethanolamine-methyl-polyethyleneglycol conjugate-2000 sodium salt (MPEG 2000 DSPE). The liposomes were prepared using thin film hydration method. The optimization of the formulation was done by employing the QbD approach. The formulation was optimized on the basis of the factors which were affecting the critical quality attributes (CQAs) such as particle size and entrapment efficiency. The final optimized formulation was characterized by assessing the particle size, percent entrapment efficiency, zeta potential, long-term stability, morphology, in vitro release and in vitro cytotoxic activity. RESULT: PEGylated liposomes having high percent entrapment efficiency (87.25 %±0.72%) could be obtained. The entrapment of drug in the liposomes was confirmed using Differential Scanning Calorimetry (DSC), Fourier Transform Infrared spectroscopy (FT-IR) and Powder X-Ray diffraction (PXRD) studies. The mean particle size of the liposomes was 113 nm±67nm and they were found to exhibit sustained release profile (56.59 %±0. 43% drug in 24h). The Small Angle Neutron Scattering (SANS) analysis revealed that the liposomes were uniform sized LUVs (nm) and were spherical in shape. The shape of the liposomes was further confirmed by transmission electron microscopy (TEM). Long term stability study indicated that the formulation was stable for three months. Sulphorhodamine B (SRB) cytotoxicity assay was carried out in HT-29 cell to prove that the PEGylated liposomal formulations had higher cytotoxicity than the conventional liposomes after 48 hours of incubation. CONCLUSION: The study affirmed that MLX loaded PEGylated liposomes had superior in vitro cytotoxicity as compared to the free drug as well as conventional liposomes. QbD resulted in the fabrication of a stable liposomal formulation with all the desirable characteristics. Hence, MLX loaded PEGylated liposomes can be considered to be a promising system for the delivery of MLX.