Proteomimetic Polymers Trigger Potent Antigen-Specific T Cell Responses to Limit Tumor Growth.

Elicitation of effective antitumor immunity following cancer vaccination requires the selective activation of distinct effector cell populations and pathways. Here we report a therapeutic approach for generating potent T cell responses using a modular vaccination platform technology capable of inducing directed immune activation, termed the Protein-like Polymer (PLP). PLPs demonstrate increased proteolytic resistance, high uptake by antigen-presenting cells (APCs), and enhanced payload-specific T cell responses. Key design parameters, namely payload linkage chemistry, degree of polymerization, and side chain composition, were varied to optimize vaccine formulations. Linking antigens to the polymer backbone using an intracellularly cleaved disulfide bond copolymerized with a diluent amount of oligo(ethylene glycol) (OEG) resulted in the highest payload-specific potentiation of antigen immunogenicity, enhancing dendritic cell (DC) activation and antigen-specific T cell responses. Vaccination with PLPs carrying either gp100, E7, or adpgk peptides significantly increased the survival of mice inoculated with B16F10, TC-1, or MC38 tumors, respectively, without the need for adjuvants. B16F10-bearing mice immunized with gp100-carrying PLPs showed increased antitumor CD8+ T cell immunity, suppressed tumor growth, and treatment synergy when paired with two distinct stimulator of interferon gene (STING) agonists. In a human papillomavirus-associated TC-1 model, combination therapy with PLP and 2'3'-cGAMP resulted in 40% of mice completely eliminating implanted tumors while also displaying curative protection from rechallenge, consistent with conferment of lasting immunological memory. Finally, PLPs can be stored long-term in a lyophilized state and are highly tunable, underscoring the unique properties of the platform for use as generalizable cancer vaccines.

Endosomal Escape of Polymer-Coated Silica Nanoparticles in Endothelial Cells.

Current investigations into hazardous nanoparticles (i.e., nanotoxicology) aim to understand the working mechanisms that drive toxicity. This understanding has been used to predict the biological impact of the nanocarriers as a function of their synthesis, material composition, and physicochemical characteristics. It is particularly critical to characterize the events that immediately follow cell stress resulting from nanoparticle internalization. While reactive oxygen species and activation of autophagy are universally recognized as mechanisms of nanotoxicity, the progression of these phenomena during cell recovery has yet to be comprehensively evaluated. Herein, primary human endothelial cells are exposed to controlled concentrations of polymer-functionalized silica nanoparticles to induce lysosomal damage and achieve cytosolic delivery. In this model, the recovery of cell functions lost following endosomal escape is primarily represented by changes in cell distribution and the subsequent partitioning of particles into dividing cells. Furthermore, multilamellar bodies are found to accumulate around the particles, demonstrating progressive endosomal escape. This work provides a set of biological parameters that can be used to assess cell stress related to nanoparticle exposure and the subsequent recovery of cell processes as a function of endosomal escape.

Tuning DNA Dissociation from Spherical Nucleic Acids for Enhanced Immunostimulation.

The stability of the core can significantly impact the therapeutic effectiveness of liposome-based drugs. While the spherical nucleic acid (SNA) architecture has elevated liposomal stability to increase therapeutic efficacy, the chemistry used to anchor the DNA to the liposome core is an underexplored design parameter with a potentially widespread biological impact. Herein, we explore the impact of SNA anchoring chemistry on immunotherapeutic function by systematically studying the importance of hydrophobic dodecane anchoring groups in attaching DNA strands to the liposome core. By deliberately modulating the size of the oligomer that defines the anchor, a library of structures has been established. These structures, combined with in vitro and in vivo immune stimulation analyses, elucidate the relationships between and importance of anchoring strength and dissociation of DNA from the SNA shell on its biological properties. Importantly, the most stable dodecane anchor, (C12)9, is superior to the n = 4-8 and 10 structures and quadruples immune stimulation compared to conventional cholesterol-anchored SNAs. When the OVA1 peptide antigen is encapsulated by the (C12)9 SNA and used as a therapeutic vaccine in an E.G7-OVA tumor model, 50% of the mice survived the initial tumor, and all of those survived tumor rechallenge. Importantly, the strong innate immune stimulation does not cause a cytokine storm compared to linear immunostimulatory DNA. Moreover, a (C12)9 SNA that encapsulates a peptide targeting SARS-CoV-2 generates a robust T cell response; T cells raised from SNA treatment kill >40% of target cells pulsed with the same peptide and ca. 45% of target cells expressing the entire spike protein. This work highlights the importance of using anchor chemistry to elevate SNA stability to achieve more potent and safer immunotherapeutics in the context of both cancer and infectious disease.

Lysyl oxidase engineered lipid nanovesicles for the treatment of triple negative breast cancer.

In the field of oncology research, a deeper understanding of tumor biology has shed light on the role of environmental conditions surrounding cancer cells. In this regard, targeting the tumor microenvironment has recently emerged as a new way to access this disease. In this work, a novel extracellular matrix (ECM)-targeting nanotherapeutic was engineered using a lipid-based nanoparticle chemically linked to an inhibitor of the ECM-related enzyme, lysyl oxidase 1 (LOX), that inhibits the crosslinking of elastin and collagen fibers. We demonstrated that, when the conjugated vesicles were loaded with the chemotherapeutic epirubicin, superior inhibition of triple negative breast cancer (TNBC) cell growth was observed both in vitro and in vivo. Moreover, in vivo results displayed prolonged survival, minimal cytotoxicity, and enhanced biocompatibility compared to free epirubicin and epirubicin-loaded nanoparticles. This all-in-one nano-based ECM-targeting chemotherapeutic may provide a key-enabling technology for the treatment of TNBC.

Electrospun anti-inflammatory patch loaded with essential oils for wound healing.

Wound healing is a complex process that consists of three overlapping phases: inflammation, proliferation, and remodeling. A bacterial infection can increase inflammation and delay this process. Microorganisms are closely related to the innate immune system, such as macrophages and neutrophils, as they can start an inflammatory cascade. Essential oils play an important role in the inhibition and prevention of bacterial growth due to their ability to reduce antimicrobial resistance. The possibility to find a strategy that combines antimicrobial and anti-inflammatory properties is particularly appealing for wound healing. In this work, we showcase a variety of patches based on electrospun polycaprolactone (PCL) nanofibers loaded with natural compounds derived from essential oils, such as thymol (THY) and tyrosol (TYR), to achieve reduced inflammation. In addition, we compared the effect these essential oils have on activated macrophages when incorporated into the PCL patch. Specifically, we demonstrate that PCL-THY resulted in more efficient down-regulation of pro-inflammatory genes related to the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κb) pathway when compared to PCL-TYR and the combination patch containing TYR and THY (i.e., PCL-TYR-THY). Furthermore, PCL-THY displayed low affinity for cell attachment, which may hinder wound adherence and integration. Overall, our results indicate that THY-loaded patches could serve as promising candidates for the fabrication of dressings that incorporate bactericidal and anti-inflammatory properties while simultaneously avoiding the limitations of traditional antibiotic-loaded devices.

Biomimetic cellular vectors for enhancing drug delivery to the lungs.

Despite recent advances in drug delivery, the targeted treatment of unhealthy cells or tissues continues to remain a priority. In cancer (much like other pathologies), delivery vectors are designed to exploit physical and biological features of unhealthy tissues that are not always homogenous across the disease. In some cases, shifting the target from unhealthy tissues to the whole organ can represent an advantage. Specifically, the natural organ-specific retention of nanotherapeutics following intravenous administration as seen in the lung, liver, and spleen can be strategically exploited to enhance drug delivery. Herein, we outline the development of a cell-based delivery system using macrophages as a delivery vehicle. When loaded with a chemotherapeutic payload (i.e., doxorubicin), these cellular vectors (CELVEC) were shown to provide continued release within the lung. This study provides proof-of-concept evidence of an alternative class of biomimetic delivery vectors that capitalize on cell size to provide therapeutic advantages for pulmonary treatments.

Electrospun Patch Functionalized with Nanoparticles Allows for Spatiotemporal Release of VEGF and PDGF-BB Promoting In Vivo Neovascularization.

The use of nanomaterials as carriers for the delivery of growth factors has been applied to a multitude of applications in tissue engineering. However, issues of toxicity, stability, and systemic effects of these platforms have yet to be fully understood, especially for cardiovascular applications. Here, we proposed a delivery system composed of poly(dl-lactide- co-glycolide) acid (PLGA) and porous silica nanoparticles (pSi) to deliver vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF). The tight spatiotemporal release of these two proteins has been proven to promote neovascularization. In order to minimize tissue toxicity, localize the release, and maintain a stable platform, we conjugated two formulations of PLGA-pSi to electrospun (ES) gelatin to create a combined ES patch releasing both PDGF and VEGF. When compared to freely dispersed particles, the ES patch cultured in vitro with neonatal cardiac cells had significantly less particle internalization (2.0 ± 1.3%) compared to free PLGA-pSi (21.5 ± 6.1) or pSi (28.7 ± 2.5) groups. Internalization was positively correlated to late-stage apoptosis with PLGA-pSi and pSi groups having increased apoptosis compared to the untreated group. When implanted subcutaneously, the ES patch was shown to have greater neovascularization than controls evidenced by increased expression of α-SMA and CD31 after 21 days. Quantitative reverse transcription-polymerase chain reaction results support increased angiogenesis by the upregulation of VEGFA, VEGFR2, vWF, and COL3A1, exhibiting a synergistic effect with the release of VEGF-A164 and PDGF-BB after 21 days in vivo. The results of this study proved that the ES patch reduced cellular toxicity and may be tailored to have a dual release of growth factors promoting localized neovascularization.

One-pot synthesis of pH-responsive hybrid nanogel particles for the intracellular delivery of small interfering RNA.

This report describes a novel, one-pot synthesis of hybrid nanoparticles formed by a nanostructured inorganic silica core and an organic pH-responsive hydrogel shell. This easy-to-perform, oil-in-water emulsion process synthesizes fluorescently-doped silica nanoparticles wrapped within a tunable coating of cationic poly(2-diethylaminoethyl methacrylate) hydrogel in one step. Transmission electron microscopy and dynamic light scattering analysis demonstrated that the hydrogel-coated nanoparticles are uniformly dispersed in the aqueous phase. The formation of covalent chemical bonds between the silica and the polymer increases the stability of the organic phase around the inorganic core as demonstrated by thermogravimetric analysis. The cationic nature of the hydrogel is responsible for the pH buffering properties of the nanostructured system and was evaluated by titration experiments. Zeta-potential analysis demonstrated that the charge of the system was reversed when transitioned from acidic to basic pH and vice versa. Consequently, small interfering RNA (siRNA) can be loaded and released in an acidic pH environment thereby enabling the hybrid particles and their payload to avoid endosomal sequestration and enzymatic degradation. These nanoparticles, loaded with specific siRNA molecules directed towards the transcript of the membrane receptor CXCR4, significantly decreased the expression of this protein in a human breast cancer cell line (i.e., MDA-MB-231). Moreover, intravenous administration of siRNA-loaded nanoparticles demonstrated a preferential accumulation at the tumor site that resulted in a reduction of CXCR4 expression.

Cell source determines the immunological impact of biomimetic nanoparticles.

Recently, engineering the surface of nanotherapeutics with biologics to provide them with superior biocompatibility and targeting towards pathological tissues has gained significant popularity. Although the functionalization of drug delivery vectors with cellular materials has been shown to provide synthetic particles with unique biological properties, these approaches may have undesirable immunological repercussions upon systemic administration. Herein, we comparatively analyzed unmodified multistage nanovectors and particles functionalized with murine and human leukocyte cellular membrane, dubbed Leukolike Vectors (LLV), and the immunological effects that may arise in vitro and in vivo. Previously, LLV demonstrated an avoidance of opsonization and phagocytosis, in addition to superior targeting of inflammation and prolonged circulation. In this work, we performed a comprehensive evaluation of the importance of the source of cellular membrane in increasing their systemic tolerance and minimizing an inflammatory response. Time-lapse microscopy revealed LLV developed using a cellular coating derived from a murine (i.e., syngeneic) source resulted in an active avoidance of uptake by macrophage cells. Additionally, LLV composed of a murine membrane were found to have decreased uptake in the liver with no significant effect on hepatic function. As biomimicry continues to develop, this work demonstrates the necessity to consider the source of biological material in the development of future drug delivery carriers.

Degradation and biocompatibility of multistage nanovectors in physiological systems.

The careful scrutiny of drug delivery systems is essential to evaluate and justify their potential for the clinic. Among the various studies necessary for preclinical testing, the impact of degradation is commonly overlooked. In this article, we investigate the effect of fabrication (porosity and nucleation layer) and environment (buffer and pH) factors on the degradation kinetics of multistage nanovectors (MSV) composed of porous silicon. The degradation by-products of MSV were exposed to endothelial cells and analyzed for detrimental effects on cellular internalization, architecture, proliferation, and cell cycle. Increases in porosity resulted in accelerated degradation exhibiting smaller-sized particles at comparable times. Removal of the nucleation layer (thin layer of small pores formed during the initial steps of etching) triggered a premature collapse of the entire central porous region of MSV. Variations in buffers prompted a faster degradation rate yielding smaller MSV within faster time frames, whereas increases in pH stimulated erosion of MSV and thus faster degradation. In addition, exposure to these degradation by-products provoked negligible impact on the proliferation and cell cycle phases on primary endothelial cells. In this study, we propose methods that lay the foundation for future investigations toward understanding the impact of the degradation of drug delivery platforms.

Synthetic nanoparticles functionalized with biomimetic leukocyte membranes possess cell-like functions.

The therapeutic efficacy of systemic drug-delivery vehicles depends on their ability to evade the immune system, cross the biological barriers of the body and localize at target tissues. White blood cells of the immune system--known as leukocytes--possess all of these properties and exert their targeting ability through cellular membrane interactions. Here, we show that nanoporous silicon particles can successfully perform all these actions when they are coated with cellular membranes purified from leukocytes. These hybrid particles, called leukolike vectors, can avoid being cleared by the immune system. Furthermore, they can communicate with endothelial cells through receptor-ligand interactions, and transport and release a payload across an inflamed reconstructed endothelium. Moreover, leukolike vectors retained their functions when injected in vivo, showing enhanced circulation time and improved accumulation in a tumour.