Thermally Robust Solvent-Free Liquid Polyplexes for Heat-Shock Protection and Long-Term Room Temperature Storage of Therapeutic Nucleic Acids.

Nucleic acid therapeutics have attracted recent attention as promising preventative solutions for a broad range of diseases. Nonviral delivery vectors, such as cationic polymers, improve the cellular uptake of nucleic acids without suffering the drawbacks of viral delivery vectors. However, these delivery systems are faced with a major challenge for worldwide deployment, as their poor thermal stability elicits the need for cold chain transportation. Here, we demonstrate a biomaterial strategy to drastically improve the thermal stability of DNA polyplexes. Importantly, we demonstrate long-term room temperature storage with a transfection efficiency maintained for at least 9 months. Additionally, extreme heat shock studies show retained luciferase expression after heat treatment at 70 °C. We therefore provide a proof of concept for a platform biotechnology that could provide long-term room temperature storage for temperature-sensitive nucleic acid therapeutics, eliminating the need for the cold chain, which in turn would reduce the cost of distributing life-saving therapeutics worldwide.

Potentiating humoral and cellular immunity using a novel hybrid polymer-lipid nanoparticle adjuvant for HBsAg-VLP vaccine.

Aluminium adjuvants are commonly used in vaccines to stimulate the immune system, but they have limited ability to promote cellular immunity which is necessary for clearing viral infections like hepatitis B. Current adjuvants that do promote cellular immunity often have undesired side effects due to the immunostimulants they contain. In this study, a hybrid polymer lipid nanoparticle (HPLNP) was developed as an efficient adjuvant for the hepatitis B surface antigen (HBsAg) virus-like particle (VLP) vaccine to potentiate both humoral and cellular immunity. The HPLNP is composed of FDA approved polyethylene glycol-b-poly (L-lactic acid) (PEG-PLLA) polymer and cationic lipid 1, 2-dioleoyl-3-trimethylammonium-propane (DOTAP), and can be easily prepared by a one-step method. The cationic optimised vaccine formulation HBsAg/HPLNP (w/w = 1/600) can maximise the cell uptake of the antigen due to the electrostatic adsorption between the vaccine nanoparticle and the cell membrane of antigen-presenting cells. The HPLNP prolonged the retention of the antigen at the injection site and enhanced the lymph node drainage of antigen, resulting in a higher concentration of serum anti-HBsAg IgG compared to the HBsAg group or the HBsAg/Al group after the boost immunisation in mice. The HPLNP also promoted a strong Th1-driven immune response, as demonstrated by the significantly improved IgG2a/IgG1 ratio, increased production of IFN-γ, and activation of CD4 + and CD8 + T cells in the spleen and lymph nodes. Importantly, the HPLNP demonstrated no systemic toxicity during immunisation. The advantages of the HPLNP, including good biocompatibility, easy preparation, low cost, and its ability to enhance both humoral and cellular immune responses, suggest its suitability as an efficient adjuvant for protein-based vaccines such as HBsAg-VLP. These findings highlight the promising potential of the HPLNP as an HBV vaccine adjuvant, offering an alternative to aluminium adjuvants currently used in vaccines.

Bioactive fatty acid analog-derived hybrid nanoparticles confer antibody-independent chemo-immunotherapy against carcinoma.

Typical chemo-immunotherapy against malignant carcinoma, is characterized by the combined application of chemotherapeutic agents and monoclonal antibodies for immune checkpoint blockade (ICB). Temporary ICB with antibodies would not depress tumor intrinsic PD-L1 expression and potential PD-L1 adaptive upregulation during chemotherapy, thus exerting limited immunotherapy efficacy. Herein, we developed novel polymer-lipid hybrid nanoparticles (2-BP/CPT-PLNs) for inducing PD-L1 degradation by inhibiting palmitoylation with bioactive palmitic acid analog 2-bromopalmitate (2-BP) to replace PD-L1 antibody (αPD-L1) for ICB therapy, thus achieving highly efficient antitumor immune via immunogenic cell death (ICD) induced by potentiated chemotherapy. GSH-responsive and biodegradable polymer-prodrug CPT-ss-PAEEP10 assisted as a cationic helper polymer could help to stabilize 2-BP/CPT-PLNs co-assembled with 2-BP, and facilitate the tumor site-specific delivery and intracellular release of water-insoluble camptothecin (CPT) in vivo. 2-BP/CPT-PLNs would reinforce cytotoxic CD8+ T cell-mediated antitumor immune response via promoting intratumoral lymphocytes cells infiltration and activation. 2-BP/CPT-PLNs significantly prevented melanoma progression and prolonged life survival of mice beyond the conventional combination of irinotecan hydrochloride (CPT-11) and αPD-L1. Our work first provided valuable instructions for developing bioactive lipid analogs-derived nanoparticles via lipid metabolism intervention for oncotherapy.

Potentiating the Immune Responses of HBsAg-VLP Vaccine Using a Polyphosphoester-Based Cationic Polymer Adjuvant.

Virus-like particle (VLP)-based vaccines are required to be associated with a suitable adjuvant to potentiate their immune responses. Herein, we report a novel, biodegradable, and biocompatible polyphosphoester-based amphiphilic cationic polymer, poly(ethylene glycol)-b-poly(aminoethyl ethylene phosphate) (PEG-PAEEP), as a Hepatitis B surface antigen (HBsAg)-VLP vaccine adjuvant. The polymer adjuvant effectively bound with HBsAg-VLP through electrostatic interactions to form a stable vaccine nanoformulation with a net positive surface charge. The nanoformulations exhibited enhanced cellular uptake by macrophages. HBsAg-VLP/PEG-PAEEP induced a significantly higher HBsAg-specific IgG titer in mice than HBsAg-VLP alone after second immunization, indicative of the antigen-dose sparing advantage of PEG-PAEEP. Furthermore, the nanoformulations exhibited a favorable biocompatibility and in vivo tolerability. This work presents the PEG-PAEEP copolymer as a promising vaccine adjuvant and as a potentially effective alternative to aluminum adjuvants.

Hybrid micelles enhance tumour therapy by remodelling biodistribution and improving intracellular drug release.

PEGylated micelles have been widely used for tumour therapy. Although PEGylation can prolong the blood circulation time, there is only less than 5% of administered micelles that can be transported to tumour sites and over 95% are cleared by the reticuloendothelial system (RES). Besides, the limited intracellular drug release also restricts their efficacy. To improve the therapeutic efficacy of PEGylated micelles, a safe, simple and efficient hybrid micellar system, composed of poly(aminoethyl ethylene phosphate)-poly(L-lactic acid) (PAEEP-PLLA) and poly(ethylene glycol)-poly(L-lactic acid) (PEG-PLLA), was developed. The hybrid micelles significantly prolonged the blood circulation time by decreasing the plasma protein adsorption and reducing the clearance by the RES. The deposition of the hybrid micelles in the liver and spleen was reduced, and the tumour accumulation was greatly improved. In addition, the intracellular drug release of the hybrid micelles was obviously increased due to the easy degradation of PAEEP in the endo/lysosomes. The tumour growth inhibition efficiency of the hybrid micelles was much higher than that of the PEG-PLLA micelles (84.5% vs. 44.5%). Furthermore, the hybrid micelles exhibited low hemolysis and reduced deposition in normal organs, which revealed their excellent bio-safety. Therefore, we established a promising hybrid micelle system for efficient anti-tumour therapy.

Multifunctional hybrid micelles with tunable active targeting and acid/phosphatase-stimulated drug release for enhanced tumor suppression.

Therapeutic efficacy of conventional single PEGylated polymeric micelles is significantly reduced by limited endocytosis and intracellular drug release. To improve drug delivery efficiency, poly (ethylene glycol)-block-poly (l-lactic acid)/(Arg-Gly-Asp-Phe)-poly (aminoethyl ethylene phosphate)-block-poly (l-lactic acid) (PEG-PLLA/RGDF-PAEEP-PLLA) hybrid micelles with tunable active targeting and acid/phosphatase-stimulated drug release are developed. The optimized hybrid micelles with 6 wt % of RGDF have favorable in vitro and in vivo activities. The hybrid micelles could temporarily shield the targeting efficacy of RGDF at pH 7.4 due to the steric effect exerted by concealment of RGDF peptides in the PEG corona, which strongly decreases the clearance by mononuclear phagocyte system and consequently improves the tumor accumulation. Inside the solid tumor with a lower acidic pH, the hybrid micelles restore the active tumor targeting property with exposed RGDF on the surface of the micelles because of the increased protonation and stretching degree of PAEEP blocks. RGDF-mediated endocytosis improves the tumor cell uptake. The hybrid micelles would also enhance intracellular drug release because of the hydrolysis of the acid/phosphatase-sensitivity of PAEEP blocks in endo/lysosome. Systemic administration of the hybrid micelles significantly inhibits tumor growth by 96% due to the integration of enhanced circulation time, tumor accumulation, cell uptake and intracellular drug release.

Self-Assembled PAEEP-PLLA Micelles with Varied Hydrophilic Block Lengths for Tumor Cell Targeting.

The properties of hydrophilic shell in micelles significantly affect the interaction between micelles and cells. Compared with frequently used polyethylene glycol (PEG) as the hydrophilic block, polyphosphoesters (PPEs) are superior in functionality, biocompatibility, and degradability. A series of amphiphilic poly(aminoethyl ethylene phosphate)/poly(l-lactide acid) (PAEEP-PLLA) copolymers were synthesized with hydrophilic PAEEP with different chain lengths. The corresponding self-assembled micelles were used for doxorubicin (Dox) entrapment. The length of hydrophilic PAEEP block on the shell affected the structure of micelles. PAEEPm-PLLA168 (m = 130 or 37) polymers formed vesicles, while PAEEPm-PLLA168 (m = 15 or 9) formed large compound micelles (LCMs), suggesting a difference in tumor cell uptake and intracellular trafficking. PAEEP15-PLLA168 polymer showed superiority on cellular uptake amount, intracellular drug release, and cell apoptosis. Lipid rafts and macropinocytosis are the leading endocytic pathways of PAEEP-PLLA micelles. The shape coupling between micelles and cell membrane facilitated cell surface features such as flattened protrusions (membrane protein) and inward-pointing hollows as well as efficient endocytosis. These results suggested that PAEEP-PLLA self-assembled block copolymer micelles may be an excellent drug delivery system for tumor treatment and that the hydrophilic chain length could regulate drug targeting to tumor cells.

Novel DiR and SPIO nanoparticles embedded PEG-PLGA nanobubbles as a multimodalimaging contrast agent.

Fluorescence dye DiR and superparamagnetic iron oxide nanoparticles (SPIONs) embedded in PEG-PLGA nanobubbles (DiR-SPIO-NBs) were produced using double emulsion method on a membrane of Shirasu porous glass (SPG). The nanobubbles encapsulated with DiR and SPIONs had a liquid core (perfluoropentane) and a PEG-PLGA shell. DiR-SPIO-NBs showed biocompatibility based on MTT cytotoxicity and hemolysis studies. The PFP encapsulated in the nanobubbles experienced phase transition under ultrasonic irradation. Nanobubbles dispersed well in saline over 3 months, and the relaxivity was 127.9 mM(-1)s(-1), suggesting that it could be used as a contrast agent in MRI. The MR and fluorescence images in vivo demonstrated that the signal intensity in the spleen and liver was significantly enhanced with the treatment of nanobubbles. In addition, results of ultrasound images suggested that the nanobubbles had persistent contrast ability. In conclusion, nanobubbles could be utilized as an US/MRI/fluorescence contrast agent.

Novel lactoferrin-conjugated amphiphilic poly(aminoethyl ethylene phosphate)/poly(L-lactide) copolymer nanobubbles for tumor-targeting ultrasonic imaging.

In the study reported here, a novel amphiphilic poly(aminoethyl ethylene phosphate)/poly(L-lactide) (PAEEP-PLLA) copolymer was synthesized by ring-opening polymerization reaction. The perfluoropentane-filled PAEEP-PLLA nanobubbles (NBs) were prepared using the O1/O2/W double-emulsion and solvent-evaporation method, with the copolymer as the shell and liquid perfluoropentane as the core of NBs. The prepared NBs were further conjugated with lactoferrin (Lf) for tumor-cell targeting. The resulting Lf-conjugated amphiphilic poly(aminoethyl ethylene phosphate)/poly(L-lactide) nanobubbles (Lf-PAEEP-PLLA NBs) were characterized by photon correlation spectroscopy, polyacrylamide gel electrophoresis, Fourier transform infrared spectroscopy, and transmission electron microscopy. The average size of the Lf-PAEEP-PLLA NBs was 328.4±5.1 nm, with polydispersity index of 0.167±0.020, and zeta potential of -12.6±0.3 mV. Transmission electron microscopy imaging showed that the Lf-PAEEP-PLLA NBs had a near-spherical structure, were quite monodisperse, and there was a clear interface between the copolymer shell and the liquid core inside the NBs. The Lf-PAEEP-PLLA NBs also exhibited good biocompatibility in cytotoxicity and hemolysis studies and good stability during storage. The high cellular uptake of Lf-PAEEP-PLLA NBs in C6 cells (low-density lipoprotein receptor-related protein 1-positive cells) at concentrations of 0-20 µg/mL indicated that the Lf provided effective targeting for brain-tumor cells. The in vitro acoustic behavior of Lf-PAEEP-PLLA NBs was evaluated using a B-mode clinical ultrasound imaging system. In vivo ultrasound imaging was performed on tumor-bearing BALB/c nude mice, and compared with SonoVue(®) microbubbles, a commercial ultrasonic contrast agent. Both in vitro and in vivo ultrasound imaging indicated that the Lf-PAEEP-PLLA NBs possessed strong, long-lasting, and tumor-enhanced ultrasonic contrast ability. Taken together, these results indicate that Lf-PAEEP-PLLA NBs represent a promising nano-sized ultrasonic contrast agent for tumor-targeting ultrasonic imaging.