Emerging advances in delivery systems for mRNA cancer vaccines.

The success of lipid nanoparticles (LNPs) in treating COVID-19 promotes further research of mRNA vaccines for cancer vaccination. Aiming at overcoming the constraints of currently available mRNA carriers, various alternative nano-vectors have been developed for delivering tumor antigen encoding mRNA and showed versatility to induce potent anti-tumor immunity. The rationally designed nano-vaccines increase the immune activation capacity of the mRNA vaccines by promoting crucial aspects including mRNA stability, cellular uptake, endosomal escape and targeting of immune cells or organs. Herein, we summarized the research progress of various mRNA based nano-vaccines that have been reported for cancer vaccination, including LNPs, lipid enveloped hybrid nanoparticles, polymeric nanoparticles etc. Several strategies that have been reported for further enhancing the immune stimulation efficacy of mRNA nano-vaccines, including developing nano-vaccines for co-delivering adjuvants, combination of immune checkpoint inhibitors, and optimizing the injection routes for boosting immune responses, have been reviewed. The progress of mRNA nano-vaccines in clinical trials and the prospect of the mRNA vaccines for cancer vaccination are also discussed.

Co-delivery of oxaliplatin prodrug liposomes with Bacillus Calmette-Guérin for chemo-immunotherapy of orthotopic bladder cancer.

To reduce recurrence rate after transurethral resection of bladder tumor, long-term intravesical instillations of Bacillus Calmette-Guérin (BCG) and/or chemotherapeutic drugs is the standard treatment for non-muscle invasive bladder carcinoma. However, the main challenges of intravesical therapy, such as short retention time and poor permeability of drugs in the bladder, often require frequent and high-dose administrations, leading to significant adverse effects and financial burden for patients. Aiming at addressing these challenges, we developed a novel approach, in which the cell-penetrating peptide modified oxaliplatin prodrug liposomes and a low-dose BCG were co-delivered via a viscous chitosan solution (LRO-BCG/CS). LRO-BCG/CS addressed these challenges by significantly improving the retention capability and permeability of chemotherapy agents across the bladder wall. Then, oxaliplatin triggered the immunogenic cell death, and the combination of BCG simultaneously further activated the systemic anti-tumor immune response in the MB49 orthotopic bladder tumor model. As a result, LRO-BCG/CS demonstrated superior anti-tumor efficacy and prolonged the survival time of tumor-bearing mice significantly, even at relatively low doses of oxaliplatin and BCG. Importantly, this combinational chemo-immunotherapy showed negligible side effects, offering a promising and well-tolerated therapeutic strategy for bladder cancer patients.

Ethanol Injection Method for Liposome Preparation.

Ethanol injection method is one of the preferred methods for liposome preparation due to its advantages including rapidity, safety, and reproducibility. This method involves the injection of phospholipid solution of ethanol into a stirred aqueous solution. Due to the diffusion of ethanol in aqueous solution, the dissolved phospholipids precipitate to form bilayer phospholipid fragments, which further fuse to form closed liposomal structures. After evaporation of ethanol, the liposomes can be finally obtained. In this chapter, we will describe the details of ethanol injection method for preparing liposomes and discuss issues that need to be considered during the fabrication process.

Smart combination of aluminum hydroxide and MF59 to induce strong cellular immune responses.

As two of the most widely used adjuvants, aluminum hydroxide and the oil-in-water emulsion MF59 have their intrinsic limitations: classical aluminum gel induces only weak cellular immune responses while MF59 cannot be used as an antigen delivery system due to its poor physical interaction with antigen molecules. Herein, we combined these two adjuvants and constructed a novel nano-vaccine delivery system by inserting aluminum hydroxide into the surface of a modified MF59 nano-emulsion (AlNEs). A model antigen ovalbumin (OVA) and an immune potentiator CpG were adsorbed on the surface of AlNEs (hereinafter AlNEs-OVA-CpG) through a facile mixing step. After subcutaneous injection, AlNEs-OVA-CpG effectively drained to lymph nodes, delivered both cargos into lymph node-resident antigen presenting cells (APCs), and escaped from lysosomes into the cytoplasm, resulting in enhanced antigen cross-presentation. Finally, AlNEs-OVA-CpG induced potent antigen-specific humoral and cellular immune responses, which significantly inhibited tumor growth and prolonged mice survival in a EG7-OVA tumor model. In sum, our results suggested that AlNEs have a great prospect to induce CD8+ T cell responses for subunit antigens.

Determination of the mechanical properties of polymeric microneedles by micromanipulation.

Precise characterization of the mechanical properties of polymeric microneedles is crucial for their successful penetration into skin and delivery of the loaded active ingredients. However, most available strategies for this purpose are based on compression of the whole patch, which only provide the average rupture force of the needles and can not give information on the variations across individual microneedles in the patch. In this study, we determined the mechanical strength of individual microneedles of two types of hyaluronic acid microneedles with or without loaded model drugs using a micromanipulation technique. The applied force as a function of displacement of the microneedles was recorded, which was used to determine the rupture displacement, rupture force, and then to derive and calculate normal stress-deformation curve, rupture stress and Young's modulus of individual microneedles. The obtained data suggest that the molecular weight of the polymer and the loading of drug into the microneedles can significantly affect the rupture behavior and mechanical properties of the microneedles, which provides a foundation for preparing sufficiently strong microneedles for controlled drug delivery.

Immunogenicity of diphtheria toxoid and poly(I:C) loaded cationic liposomes after hollow microneedle-mediated intradermal injection in mice.

In this study, we aimed to investigate the immunogenicity of cationic liposomes loaded with diphtheria toxoid (DT) and poly(I:C) after hollow microneedle-mediated intradermal vaccination in mice. The following liposomal formulations were studied: DT loaded liposomes, a mixture of free DT and poly(I:C)-loaded liposomes, a mixture of DT-loaded liposomes and free poly(I:C), and liposomal formulations with DT and poly(I:C) either individually or co-encapsulated in the liposomes. Reference groups were DT solution adjuvanted with or without poly(I:C) (DT/poly(I:C)). The liposomal formulations were characterized in terms of particle size, zeta potential, loading and release of DT and poly(I:C). After intradermal injection of BALB/c mice with the formulations through a hollow microneedle, the immunogenicity was assessed by DT-specific ELISAs. All formulations induced similar total IgG and IgG1 titers. However, all the liposomal groups containing both DT and poly(I:C) showed enhanced IgG2a titers compared to DT/poly(I:C) solution, indicating that the immune response was skewed towards a Th1 direction. This enhancement was similar for all liposomal groups that contain both DT and poly(I:C) in the formulations. Our results reveal that a mixture of DT encapsulated liposomes and poly(I:C) encapsulated liposomes have a similar effect on the antibody responses as DT and poly(I:C) co-encapsulated liposomes. These findings may have implications for future design of liposomal vaccine delivery systems.

Development of PLGA nanoparticle loaded dissolving microneedles and comparison with hollow microneedles in intradermal vaccine delivery.

Skin is an attractive but also very challenging immunisation site for particulate subunit vaccines. The aim of this study was to develop hyaluronan (HA)-based dissolving microneedles (MNs) loaded with PLGA nanoparticles (NPs) co-encapsulating ovalbumin (OVA) and poly(I:C) for intradermal immunisation. The NP:HA ratio used for the preparation of dissolving MNs appeared to be critical for the quality of MNs and their dissolution in ex vivo human skin. Asymmetrical flow field-flow fractionation and dynamic light scattering were used to analyse the NPs released from the MNs in vitro. Successful release of the NPs depended on the drying conditions during MN preparation. The delivered antigen dose from dissolving MNs in mice was determined to be 1 µg OVA, in NPs or as free antigen, by using near-infrared fluorescence imaging. Finally, the immunogenicity of the NPs after administration of dissolving MNs (NP:HA weight ratio 1:4) was compared with that of hollow MN-delivered NPs in mice. Immunization with free antigen in dissolving MNs resulted in equally strong immune responses compared to delivery by hollow MNs. However, humoral and cellular immune responses evoked by NP-loaded dissolving MNs were inferior to those elicited by NPs delivered through a hollow MN. In conclusion, we identified several critical formulation parameters for the further development of NP-loaded dissolving MNs.

Hollow microneedle-mediated intradermal delivery of model vaccine antigen-loaded PLGA nanoparticles elicits protective T cell-mediated immunity to an intracellular bacterium.

The skin is an attractive organ for immunization due to the presence of a large number of epidermal and dermal antigen-presenting cells. Hollow microneedles allow for precise and non-invasive intradermal delivery of vaccines. In this study, ovalbumin (OVA)-loaded poly(lactic-co-glycolic acid) (PLGA) nanoparticles with and without TLR3 agonist poly(I:C) were prepared and administered intradermally by hollow microneedles. The capacity of the PLGA nanoparticles to induce a cytotoxic T cell response, contributing to protection against intracellular pathogens, was examined. We show that a single injection of OVA-loaded PLGA nanoparticles, compared to soluble OVA, primed both adoptively transferred antigen-specific naïve transgenic CD8+ and CD4+ T cells with markedly high efficiency. Applying a triple immunization protocol, PLGA nanoparticles primed also endogenous OVA-specific CD8+ T cells. Immune response, following immunization with in particular anionic PLGA nanoparticles co-encapsulated with OVA and poly(I:C), provided protection against a recombinant strain of the intracellular bacterium Listeria monocytogenes, secreting OVA. Taken together, we show that PLGA nanoparticle formulation is an excellent delivery system for protein antigen into the skin and that protective cellular immune responses can be induced using hollow microneedles for intradermal immunizations.

Intradermal vaccination with hollow microneedles: A comparative study of various protein antigen and adjuvant encapsulated nanoparticles.

In this study, we investigated the potential of intradermal delivery of nanoparticulate vaccines to modulate the immune response of protein antigen using hollow microneedles. Four types of nanoparticles covering a broad range of physiochemical parameters, namely poly (lactic-co-glycolic) (PLGA) nanoparticles, liposomes, mesoporous silica nanoparticles (MSNs) and gelatin nanoparticles (GNPs) were compared. The developed nanoparticles were loaded with a model antigen (ovalbumin (OVA)) with and without an adjuvant (poly(I:C)), followed by the characterization of size, zeta potential, morphology, and loading and release of antigen and adjuvant. An in-house developed hollow-microneedle applicator was used to inject nanoparticle suspensions precisely into murine skin at a depth of about 120µm. OVA/poly(I:C)-loaded nanoparticles and OVA/poly(I:C) solution elicited similarly strong total IgG and IgG1 responses. However, the co-encapsulation of OVA and poly(I:C) in nanoparticles significantly increased the IgG2a response compared to OVA/poly(I:C) solution. PLGA nanoparticles and liposomes induced stronger IgG2a responses than MSNs and GNPs, correlating with sustained release of the antigen and adjuvant and a smaller nanoparticle size. When examining cellular responses, the highest CD8+ and CD4+ T cell responses were induced by OVA/poly(I:C)-loaded liposomes. In conclusion, the applicator controlled hollow microneedle delivery is an excellent method for intradermal injection of nanoparticle vaccines, allowing selection of optimal nanoparticle formulations for humoral and cellular immune responses.

Encapsulation of azithromycin into polymeric microspheres by reduced pressure-solvent evaporation method.

Azithromycin loaded microspheres with blends of poly-l-lactide and ploy-D,L-lactide-co-glycolide as matrices were prepared by the atmosphere-solvent evaporation (ASE) and reduced pressure-solvent evaporation (RSE) method. Both the X-ray diffraction spectra and DSC thermographs demonstrated that poly-L-lactide existed in a crystalline form in the ASE microspheres, while an amorphous form was present in the RSE formulations. Besides, solvent removal at atmosphere gave microspheres of porous and rough surfaces, but smooth surfaces appeared in the RSE microspheres. The incorporation efficiency as well as the burst release (cumulative release in the first 24h) in the ASE formulations was 39.94 ± 1.18% and 23.96 ± 2.01% respectively, yet the encapsulation efficiency of the microspheres fabricated under 385 mmHg was high up to 57.19 ± 3.81% and the burst release was 4.12 ± 0.15%. The in vitro drug release studies indicated that the ASE microspheres presented a zero-order profile; while the RSE formulations followed first-order kinetics. Other factors including solidification time, temperature, drug to polymer ratio and pH value of the continuous phase could also influence the physicochemical characteristics and release profiles of microspheres. In conclusion, the overall improvement of microspheres in appearance, encapsulation efficiency and controlled drug release through the RSE method could be easily fulfilled under optimal preparation conditions.