Priming with oncolytic adenovirus followed by anti-PD-1 and paclitaxel treatment leads to improved anti-cancer efficacy in the 3D TNBC model.

Triple-negative breast cancer (TNBC) is considered one of the most incurable malignancies due to its clinical characteristics, including high invasiveness, high metastatic potential, proneness to relapse, and poor prognosis. Therefore, it remains a critical unmet medical need. On the other hand, poor delivery efficiency continues to reduce the efficacy of anti-cancer therapeutics developed against solid tumours using various strategies, such as genetically engineered oncolytic vectors used as nanocarriers. The study was designed to evaluate the anti-tumour efficacy of a novel combinatorial therapy based on oncolytic adenovirus AdV5/3-D24-ICOSL-CD40L with an anti-PD-1 (pembrolizumab) and paclitaxel (PTX). Here, we first tested the antineoplastic effect in two-dimensional (2D) and three-dimensional (3D) breast cancer models in MDA-MB-231, MDA-MB-468 and MCF-7 cells. Then, to further evaluate the efficacy of combinatorial therapy, including immunological aspects, we established a three-dimensional (3D) co-culture model based on MDA-MB-231 cells with peripheral blood mononuclear cells (PBMCs) to create an integrated system that more closely mimics the complexity of the tumour microenvironment and interacts with the immune system. Treatment with OV as a priming agent, followed by pembrolizumab and then paclitaxel, was the most effective in reducing the tumour volume in TNBC co-cultured spheroids. Further, T-cell phenotyping analyses revealed significantly increased infiltration of CD8+, CD4+ T and Tregs cells. Moreover, the observed anti-tumour effects positively correlated with the level of CD4+ T cell infiltrates, suggesting the development of anti-cancer immunity. Our study demonstrated that combining different immunotherapeutic agents (virus, pembrolizumab) with PTX reduced the tumour volume of the TNBC co-cultured spheroids compared to relevant controls. Importantly, sequential administration of the investigational agents (priming with the vector) further enhanced the anti-cancer efficacy in 3D culture over other groups tested. Taken together, these results support further evaluation of the virus in combination with anti-PD-1 and PTX for the treatment of triple-negative breast cancer patients. Importantly, further studies with in vivo models should be conducted to better understand the translational aspects of tested therapy.

In situ administration of STING-activating hyaluronic acid conjugate primes anti-glioblastoma immune response.

Glioblastoma (GBM) is an aggressive brain tumor, with a highly immunosuppressive tumor immune microenvironment (TIME). In this work, we investigated the use of the STimulator of INterferon Genes (STING) pathway as an effective means to remodel the GBM TIME through the recruitment of both innate and adaptive immune cell populations. Using hyaluronic acid (HA), we developed a novel polymer-drug conjugate of a non-nucleotide STING agonist (MSA2), called HA-MSA2 for the in situ treatment of GBM. In JAWSII cells, HA-MSA2 exerted a greater increase of STING signaling and upregulation of STING-related downstream cyto-/chemokines in immune cells than the free drug. HA-MSA2 also elicited cancer cell-intrinsic immunostimulatory gene expression and promoted immunogenic cell death of GBM cells. In the SB28 GBM model, local delivery of HA-MSA2 induced a delay in tumor growth and a significant extension of survival. The analysis of the TIME showed a profound shift in the GBM immune landscape after HA-MSA2 treatment, with higher infiltration by innate and adaptive immune cells including dendritic, natural killer (NK) and CD8 T cell populations. The therapeutic potential of this novel polymer conjugate warrants further investigation, particularly with other chemo-immunotherapeutics or cancer vaccines as a promising combinatorial therapeutic approach.

Local delivery of doxorubicin prodrug via lipid nanocapsule-based hydrogel for the treatment of glioblastoma.

Glioblastoma (GBM) recurrences appear in most cases around the resection cavity borders and arise from residual GBM cells that cannot be removed by surgery. Here, we propose a novel treatment that combines the advantages of nanomedicine and local drug delivery to target these infiltrating GBM cells. We developed an injectable lipid nanocapsule (LNC)-based formulation loaded with lauroyl-doxorubicin prodrug (DOXC12). Firstly, we demonstrated the efficacy of intratumoral administration of DOXC12 in GL261 GBM-bearing mice, which extended mouse survival. Then, we formulated an injectable hydrogel by mixing the appropriate amount of prodrug with the lipophilic components of LNC. We optimized the hydrogel by incorporating cytidine-C16 (CytC16) to achieve a mechanical stiffness adapted for an application in the brain post-surgery (DOXC12-LNCCL). DOXC12-LNCCL exhibited high DOXC12 encapsulation efficiency (95%) and a size of approximately 60 nm with sustained drug release for over 1 month in vitro. DOXC12-LNCCL exhibited enhanced cytotoxicity compared to free DOXC12 (IC50 of 349 and 86 nM, respectively) on GL261 GBM cells and prevented the growth of GL261 spheroids cultured on organotypic brain slices. In vivo, post-surgical treatment with DOXC12-LNCCL significantly improved the survival of GL261-bearing mice. The combination of this local treatment with the systemic administration of anti-inflammatory drug ibuprofen further delayed the onset of recurrences. In conclusion, our study presents a promising therapeutic approach for the treatment of GBM. By targeting residual GBM cells and reducing the inflammation post-surgery, we present a new strategy to delay the onset of recurrences in the gap period between surgery and standard of care therapy.

Control of cell penetration enhancer shielding and endosomal escape-kinetics crucial for efficient and biocompatible siRNA delivery.

Although cationic liposomes are efficient carriers for nucleic acid delivery, their toxicity often hampers the clinical translation. Polyethylene glycol (PEG) coating has been largely used to improve their stability and reduce toxicity. Nevertheless, it has been found to decrease the transfection process. In order to exploit the advantages of cationic liposomes and PEG decoration for nucleic acid delivery, liposomes decorated with tetraArg-[G-1]-distearoyl glycerol (Arg4-DAG) dendronic oligo-cationic lipid enhancer (OCE) and PEG-lipid have been investigated. Non decorated or OCE-decorated lipoplexes (OCEfree-LPX and OCE-LPX, respectively) were obtained by lipid film hydration using oligonucleotide (ON) solutions. PEG and OCE/PEG decorated lipoplexes (PEG-OCEfree-LPX and PEG-OCE-LPX, respectively) were obtained by post-insertion of 2 or 5 kDa PEG-DSPE on preformed lipoplexes. The OCE decoration yielded lipoplexes with size of about 240 nm, 84% loading efficiency at 10 N/P ratio, ten times higher than OCEfree-LPX, and prevented the ON release when incubated with physiological heparin concentration or with plasma. The PEG decoration reduced the zeta potential, enhanced the lipoplex stability in serum and decreased both hemolysis and cytotoxicity, while it did not affect the lipoplex size and ON loading. With respect to OCEfree-LPX, the OCE-LPX remarkably associated with cells and were taken up by different cancer cell lines (HeLa and MDA-MB-231). Interestingly, 2 or 5 kDa PEG decoration did not reduce either the cell interaction or the cell up-take of the cationic lipoplexes. With siRNA as a payload, OCE enabled efficient internalization, but endosomal release was hampered. Post-transfection treatment with the lysosomotropic drug chloroquine allowed to identify the optimal time point for endosomal escape. Chloroquine treatment after 12 to 20 h of LPX pre-incubation enabled siRNA mediated target knockdown indicating that this is the time window of endo-lysosomal processing. This indicates that OCE can protect siRNA from lysosomal degradation for up to 20 h, as shown by these rescue experiments.

Hyaluronic acid-antigens conjugates trigger potent immune response in both prophylactic and therapeutic immunization in a melanoma model.

Immunotherapy of advanced melanoma has encountered significant hurdles in terms of clinical efficacy. Here, we designed a clinically translatable hyaluronic acid (HA)-based vaccine delivering a combination of major histocompatibility complex (MHC) class I- and class II-restricted melanoma antigens (TRP2 and Gp100, respectively) conjugated to HA. HA-nanovaccine (HA-TRP2-Gp100 conjugate) exhibited tropism in the lymph nodes and promoted stimulation of the immune response (2.3-fold higher than the HA+TRP2+Gp100). HA-nanovaccine significantly delayed the growth of B16F10 melanoma and extended survival in both the prophylactic and therapeutic settings (median survival of 22 and 27, respectively, vs 17 days of the untreated group). Moreover, mice prophylactically treated with the HA-nanovaccine displayed significantly higher CD8+ and CD4+ T-cell/Treg ratios in both the spleen and tumor at day 16, suggesting that the HA-nanovaccine overcame the immunosuppressive tumor microenvironment. Superior infiltration of active CD4+ and CD8+ T cells was observed at the endpoint. This study supports the conclusion that HA potentiates the effect of a combination of MHC I and MHC II antigens via a potent immune response against melanoma.

Combination of local immunogenic cell death-inducing chemotherapy and DNA vaccine increases the survival of glioblastoma-bearing mice.

Immunotherapy efficacy as monotherapy is negligible for glioblastoma (GBM). We hypothesized that combining therapeutic vaccination using a plasmid encoding an epitope derived from GBM-associated antigen (pTOP) with local delivery of immunogenic chemotherapy using mitoxantrone-loaded PEGylated PLGA-based nanoparticles (NP-MTX) would improve the survival of GBM-bearing mice by stimulating an antitumor immune response. We first proved that MTX retained its ability to induce cytotoxicity and immunogenic cell death of GBM cells after encapsulation. Intratumoral delivery of MTX or NP-MTX increased the frequency of IFN-γ-secreting CD8 T cells. NP-MTX mixed with free MTX in combination with pTOP DNA vaccine increased the median survival of GL261-bearing mice and increased M1-like macrophages in the brain. The addition of CpG to this combination abolished the survival benefit but led to increased M1 to M2 macrophage ratio and IFN-γ-secreting CD4 T cell frequency. These results highlight the benefits of combination strategies to potentiate immunotherapy and improve GBM outcome.

Influence of Folate-Targeted Gold Nanoparticles on Subcellular Localization and Distribution into Lysosomes.

The cell interaction, mechanism of cell entry and intracellular fate of surface decorated nanoparticles are known to be affected by the surface density of targeting agents. However, the correlation between nanoparticles multivalency and kinetics of the cell uptake process and disposition of intracellular compartments is complicated and dependent on a number of physicochemical and biological parameters, including the ligand, nanoparticle composition and colloidal properties, features of targeted cells, etc. Here, we have carried out an in-depth investigation on the impact of increasing folic acid density on the kinetic uptake process and endocytic route of folate (FA)-targeted fluorescently labelled gold nanoparticles (AuNPs). A set of AuNPs (15 nm mean size) produced by the Turkevich method was decorated with 0-100 FA-PEG3.5kDa-SH molecules/particle, and the surface was saturated with about 500 rhodamine-PEG2kDa-SH fluorescent probes. In vitro studies carried out using folate receptor overexpressing KB cells (KBFR-high) showed that the cell internalization progressively increased with the ligand surface density, reaching a plateau at 50:1 FA-PEG3.5kDa-SH/particle ratio. Pulse-chase experiments showed that higher FA density (50 FA-PEG3.5kDa-SH molecules/particle) induces more efficient particle internalization and trafficking to lysosomes, reaching the maximum concentration in lysosomes at 2 h, than the lower FA density of 10 FA-PEG3.5kDa-SH molecules/particle. Pharmacological inhibition of endocytic pathways and TEM analysis showed that particles with high folate density are internalized predominantly by a clathrin-independent process.

Stimuli-Responsive Multifunctional Nanomedicine for Enhanced Glioblastoma Chemotherapy Augments Multistage Blood-to-Brain Trafficking and Tumor Targeting.

Minimal therapeutic advances have been achieved over the past two decades for glioblastoma (GBM), which remains an unmet clinical need. Here, hypothesis-driven stimuli-responsive nanoparticles (NPs) for docetaxel (DTX) delivery to GBM are reported, with multifunctional features that circumvent insufficient blood-brain barrier (BBB) trafficking and lack of GBM targeting-two major hurdles for anti-GBM therapies. NPs are dual-surface tailored with a i) brain-targeted acid-responsive Angiopep-2 moiety that triggers NP structural rearrangement within BBB endosomal vesicles, and ii) L-Histidine moiety that provides NP preferential accumulation into GBM cells post-BBB crossing. In tumor invasive margin patient cells, the stimuli-responsive multifunctional NPs target GBM cells, enhance cell uptake by 12-fold, and induce three times higher cytotoxicity in 2D and 3D cell models. Moreover, the in vitro BBB permeability is increased by threefold. A biodistribution in vivo trial confirms a threefold enhancement of NP accumulation into the brain. Last, the in vivo antitumor efficacy is validated in GBM orthotopic models following intratumoral and intravenous administration. Median survival and number of long-term survivors are increased by 50%. Altogether, a preclinical proof of concept supports these stimuli-responsive multifunctional NPs as an effective anti-GBM multistage chemotherapeutic strategy, with ability to respond to multiple fronts of the GBM microenvironment.

Synergistic effect of doxorubicin lauroyl hydrazone derivative delivered by α-tocopherol succinate micelles for the treatment of glioblastoma.

We hypothesized that tocopherol succinate (TOS) and D-α-tocopherol polyethylene2000 succinate (TPGS2000) micelles could work as a drug delivery system while enhancing the anti-cancer efficacy of doxorubicin lauryl hydrazone derivative (DOXC12) for the treatment of glioblastoma. The DOXC12-TOS-TPGS2000 micelles were formulated with synthesized DOXC12 and TPGS2000. They showed a high drug loading of hydrophobic DOXC12 (29%), a size of <100 nm and a pH sensitive drug release behaviour. In vitro, fast uptake of DOXC12-TOS-TPGS2000 micelles by GL261 cells was observed. For cytotoxicity, DOXC12-TOS-TPGS2000 micelles were evaluated on two glioblastoma cell lines and showed synergism between DOXC12 and TOS-TPGS2000. The higher cytotoxicity of DOXC12-TOS-TPGS2000 micelles was mainly caused by necrosis. The DOXC12-TOS-TPGS2000 micelles seem to be a promising delivery system for enhancing the anticancer efficacy of doxorubicin in glioblastoma (GBM).

Tailoring surface properties of liposomes for dexamethasone intraocular administration.

Diseases of the posterior eye segment are often characterized by intraocular inflammation, which causes, in the long term, severe impairment of eye functions and, ultimately, vision loss. Aimed at enhancing the delivery of anti-inflammatory drugs to the posterior eye segment upon intravitreal administration, we developed liposomes with an engineered surface to control their diffusivity in the vitreous and retina association. Hydrogenated soybean phosphatidylcholine (HSPC)/cholesterol liposomes were coated with (agmatinyl)6-maltotriosyl-acetamido-N-(octadec-9-en-1-yl)hexanamide (Agm6-M-Oleate), a synthetic non-peptidic cell penetration enhancer (CPE), and/or 5% of mPEG2kDa-DSPE. The zeta potential of liposomes increased, and the mobility in bovine vitreous and colloidal stability decreased with the Agm6-M-Oleate coating concentration. Oppositely, mPEG2kDa-DSPE decreased the zeta potential of liposomes and restored both the diffusivity and the stability in vitreous. Liposomes with 5 mol% Agm6-M-Oleate coating were well tolerated by ARPE-19 retina cells either with or without mPEG2kDa-DSPE, while 10 mol% Agm6-M-Oleate showed cytotoxicity. Agm6-M-Oleate promoted the association of liposomes to ARPE-19 cells with respect to plain liposomes, while mPEG2kDa-DSPE slightly reduced the cell interaction. Dexamethasone hemisuccinate (DH) was remotely loaded into liposomes with a loading capacity of ∼10 wt/wt%. Interestingly, mPEG2kDa-DSPE coating reduced the rate of DH release and enhanced the disposition of Agm6-M-Oleate coated liposomes in the ARPE-19 cell cytosol resulting in a more efficient anti-inflammatory effect. Finally, mPEG2kDa-DSPE enhanced the association of DH-loaded Agm6-M-Oleate coated liposomes to explanted rat retina, which reflected in higher viability of inner and outer nuclear layer cells.

Combination of hyaluronic acid conjugates with immunogenic cell death inducer and CpG for glioblastoma local chemo-immunotherapy elicits an immune response and induces long-term survival.

The efficacy of standard glioblastoma (GBM) treatments has been limited due to the highly immunosuppressive tumor immune microenvironment, interpatient tumor heterogenicity and anatomical barriers, such as the blood brain barrier. In the present work, we hypothesized that a new local therapy based on the combination of doxorubicin (DOX) as an immunogenic cell death (ICD) inducer and CpG, a Toll-like receptor (TLR)-9 agonist, would act synergistically to eradicate GBM. DOX and CpG were first tested in an orthotopic GL261 GBM model showing enhanced survival. To improve the outcome with a reduced dose, we designed bioresponsive hyaluronic acid (HA)-drug conjugates for effective in situ chemoimmunotherapy. HA was derivatized with CpG. The new HA-CpG conjugate showed high efficacy in re-educating protumoral M2-like microglia into an antitumoral M1-like phenotype, inducing the expression of immune-stimulatory cytokines. DOX was also conjugated to HA. DOX conjugation increased ICD induction in GL261 cells. Finally, a combination of the conjugates was explored in an orthotopic GL261 GBM model. The local delivery of combined HA-DOX + HA-CpG into the tumor mass elicited antitumor CD8+ T cell responses in the brain tumor microenvironment and reduced the infiltration of M2-like tumor-associated macrophages and myeloid-derived suppressor cells. Importantly, the combination of HA-DOX and HA-CpG induced long-term survival in >66% of GBM-bearing animals than other treatments (no long-term survivor observed), demonstrating the benefits of conjugating synergistic drugs to HA nanocarrier. These results emphasize that HA-drug conjugates constitute an effective drug delivery platform for local chemoimmunotherapy against GBM and open new perspectives for the treatment of other brain cancers and brain metastasis.

Combination of DNA Vaccine and Immune Checkpoint Blockades Improves the Immune Response in an Orthotopic Unresectable Glioblastoma Model.

Combination immunotherapy has emerged as a promising strategy to increase the immune response in glioblastoma (GBM) and overcome the complex immunosuppression occurring in its microenvironment. In this study, we hypothesized that combining DNA vaccines-to stimulate a specific immune response-and dual immune checkpoint blockade (ICB)-to decrease the immunosuppression exerted on T cells-will improve the immune response and the survival in an orthotopic unresectable GL261 model. We first highlighted the influence of the insertion position of a GBM epitope sequence in a plasmid DNA vaccine encoding a vesicular stomatitis virus glycoprotein (VSV-G) (here referred to as pTOP) in the generation of a specific and significant IFN-γ response against the GBM antigen TRP2 by inserting a CD8 epitope sequence in specific permissive sites. Then, we combined the pTOP vaccine with anti-PD-1 and anti-CTLA-4 ICBs. Immune cell analysis revealed an increase in effector T cell to Treg ratios in the spleens and an increase in infiltrated IFN-γ-secreting CD8 T cell frequency in the brains following combination therapy. Even if the survival was not significantly different between dual ICB and combination therapy, we offer a new immunotherapeutic perspective by improving the immune landscape in an orthotopic unresectable GBM model.

Design of Bio-Responsive Hyaluronic Acid-Doxorubicin Conjugates for the Local Treatment of Glioblastoma.

Glioblastoma is an unmet clinical need. Local treatment strategies offer advantages, such as the possibility to bypass the blood-brain barrier, achieving high drug concentrations at the glioblastoma site, and consequently reducing systemic toxicity. In this study, we evaluated the feasibility of using hyaluronic acid (HA) for the local treatment of glioblastoma. HA was conjugated to doxorubicin (DOX) with distinct bio-responsive linkers (direct amide conjugation HA-NH-DOX), direct hydrazone conjugation (HA-Hz-DOX), and adipic hydrazone (HA-AdpHz-DOX). All HA-DOX conjugates displayed a small size (less than 30 nm), suitable for brain diffusion. HA-Hz-DOX showed the best performance in killing GBM cells in both 2D and 3D in vitro models and displayed superior activity in a subcutaneous GL261 tumor model in vivo compared to free DOX and other HA-DOX conjugates. Altogether, these results demonstrate the feasibility of HA as a polymeric platform for the local treatment of glioblastoma and the importance of rationally designing conjugates.

Immunotherapy for glioblastoma: the promise of combination strategies.

Glioblastoma (GBM) treatment has remained almost unchanged for more than 20 years. The current standard of care involves surgical resection (if possible) followed by concomitant radiotherapy and chemotherapy. In recent years, immunotherapy strategies have revolutionized the treatment of many cancers, increasing the hope for GBM therapy. However, mostly due to the high, multifactorial immunosuppression occurring in the microenvironment, the poor knowledge of the neuroimmune system and the presence of the blood-brain barrier, the efficacy of immunotherapy in GBM is still low. Recently, new strategies for GBM treatments have employed immunotherapy combinations and have provided encouraging results in both preclinical and clinical studies. The lessons learned from clinical trials highlight the importance of tackling different arms of immunity. In this review, we aim to summarize the preclinical evidence regarding combination immunotherapy in terms of immune and survival benefits for GBM management. The outcomes of recent studies assessing the combination of different classes of immunotherapeutic agents (e.g., immune checkpoint blockade and vaccines) will be discussed. Finally, future strategies to ameliorate the efficacy of immunotherapy and facilitate clinical translation will be provided to address the unmet medical needs of GBM.

Depletion of Mannose Receptor-Positive Tumor-associated Macrophages via a Peptide-targeted Star-shaped Polyglutamate Inhibits Breast Cancer Progression in Mice.

Although many studies have explored the depletion of tumor-associated macrophages (TAM) as a therapeutic strategy for solid tumors, currently available compounds suffer from poor efficacy and dose-limiting side effects. Here, we developed a novel TAM-depleting agent ("OximUNO") that specifically targets CD206+ TAMs and demonstrated efficacy in a triple-negative breast cancer (TNBC) mouse model. OximUNO comprises a star-shaped polyglutamate (St-PGA) decorated with the CD206-targeting peptide mUNO that carries the chemotherapeutic drug doxorubicin (DOX). In the TNBC model, a fluorescently labeled mUNO-decorated St-PGA homed to CD206+ TAMs within primary lesions and metastases. OximUNO exhibited no acute liver or kidney toxicity in vivo. Treatment with OximUNO reduced the progression of primary tumor lesions and pulmonary metastases, significantly diminished the number of CD206+ TAMs and increased the CD8/FOXP3 expression ratio (indicating immunomodulation). Our findings suggest the potential benefit of OximUNO as a TAM-depleting agent for TNBC treatment. Importantly, our studies also represent a novel design of a peptide-targeted St-PGA as a targeted therapeutic nanoconjugate. Significance: A peptide-targeted nanoformulation of DOX exclusively eliminates mannose receptor+ TAMs in breast cancer models, generating response without off-target effects (a drawback of many TAM-depleting agents under clinical study).

Rationally designed drug delivery systems for the local treatment of resected glioblastoma.

Glioblastoma (GBM) is a particularly aggressive brain cancer associated with high recurrence and poor prognosis. The standard of care, surgical resection followed by concomitant radio- and chemotherapy, leads to low survival rates. The local delivery of active agents within the tumor resection cavity has emerged as an attractive means to initiate oncological treatment immediately post-surgery. This complementary approach bypasses the blood-brain barrier, increases the local concentration at the tumor site while reducing or avoiding systemic side effects. This review will provide a global overview on the local treatment for GBM with an emphasis on the lessons learned from past clinical trials. The main parameters to be considered to rationally design fit-of-purpose biomaterials and develop drug delivery systems for local administration in the GBM resection cavity to prevent the tumor recurrence will be described. The intracavitary local treatment of GBM should i) use materials that facilitate translation to the clinic; ii) be characterized by easy GMP effective scaling up and easy-handling application by the neurosurgeons; iii) be adaptable to fill the tumor-resected niche, mold to the resection cavity or adhere to the exposed brain parenchyma; iv) be biocompatible and possess mechanical properties compatible with the brain; v) deliver a therapeutic dose of rationally-designed or repurposed drug compound(s) into the GBM infiltrative margin. Proof of concept with high translational potential will be provided. Finally, future perspectives to facilitate the clinical translation of the local perisurgical treatment of GBM will be discussed.

Polymer Coated Oncolytic Adenovirus to Selectively Target Hepatocellular Carcinoma Cells.

Despite significant advances in chemotherapy, the overall prognosis of hepatocellular carcinoma (HCC) remains extremely poor. HCC targeting strategies were combined with the tumor cell cytotoxicity of oncolytic viruses (OVs) to develop a more efficient and selective therapeutic system. OVs were coated with a polygalactosyl-b-agmatyl diblock copolymer (Gal32-b-Agm29), with high affinity for the asialoglycoprotein receptor (ASGPR) expressed on the liver cell surface, exploiting the electrostatic interaction of the positively charged agmatine block with the negatively charged adenoviral capsid surface. The polymer coating altered the viral particle diameter (from 192 to 287 nm) and zeta-potential (from -24.7 to 23.3 mV) while hiding the peculiar icosahedral symmetrical OV structure, as observed by TEM. Coated OVs showed high potential therapeutic value on the human hepatoma cell line HepG2 (cytotoxicity of 72.4% ± 4.96), expressing a high level of ASGPRs, while a lower effect was attained with ASPGR-negative A549 cell line (cytotoxicity of 54.4% ± 1.59). Conversely, naked OVs showed very similar effects in both tested cell lines. Gal32-b-Agm29 OV coating enhanced the infectivity and immunogenic cell death program in HepG2 cells as compared to the naked OV. This strategy provides a rationale for future studies utilizing oncolytic viruses complexed with polymers toward effective treatment of hepatocellular carcinoma.

Lipid-Polyglutamate Nanoparticle Vaccine Platform.

Peptide-based subunit vaccines are attractive in view of personalized cancer vaccination with neo-antigens, as well as for the design of the newest generation of vaccines against infectious diseases. Key to mounting robust antigen-specific immunity is delivery of antigen to antigen-presenting (innate immune) cells in lymphoid tissue with concomitant innate immune activation to promote antigen presentation to T cells and to shape the amplitude and nature of the immune response. Nanoparticles that co-deliver both peptide antigen and molecular adjuvants are well suited for this task. However, in the context of peptide-based antigen, an unmet need exists for a generic strategy that allows for co-encapsulation of peptide and molecular adjuvants due to the stark variation in physicochemical properties based on the amino acid sequence of the peptide. These properties also strongly differ from those of many molecular adjuvants. Here, we devise a lipid nanoparticle (LNP) platform that addresses these issues. Key in our concept is poly(l-glutamic acid) (PGA), which serves as a hydrophilic backbone for conjugation of, respectively, peptide antigen (Ag) and an imidazoquinoline (IMDQ) TLR7/8 agonist as a molecular adjuvant. Making use of the PGA's polyanionic nature, we condensate PGA-Ag and PGA-IMDQ into LNP by electrostatic interaction with an ionizable lipid. We show in vitro and in vivo in mouse models that LNP encapsulation favors uptake by innate immune cells in lymphoid tissue and promotes the induction of Ag-specific T cells responses both after subcutaneous and intravenous administration.

Glabrescione B delivery by self-assembling micelles efficiently inhibits tumor growth in preclinical models of Hedgehog-dependent medulloblastoma.

Aberrant activation of the Hedgehog (Hh) pathway leads to the development of several tumors, including medulloblastoma (MB), the most common pediatric brain malignancy. Hh inhibitors acting on GLI1, the final effector of Hh signaling, offer a valuable opportunity to overcome the pitfalls of the existing therapies to treat Hh-driven cancers. In this study, the toxicity, delivery, biodistribution, and anticancer efficacy of Glabrescione B (GlaB), a selective GLI1 inhibitor, were investigated in preclinical models of Hh-dependent MB. To overcome its poor water solubility, GlaB was formulated with a self-assembling amphiphilic polymer forming micelles, called mPEG5kDa-cholane. mPEG5kDa-cholane/GlaB showed high drug loading and stability, low cytotoxicity, and long permanence in the bloodstream. We found that mPEG5kDa-cholane efficiently enhanced the solubility of GlaB, thus avoiding the use of organic solvents. mPEG5kDa-cholane/GlaB possesses favorable pharmacokinetics and negligible toxicity. Remarkably, GlaB encapsulated in mPEG5kDa-cholane micelles was delivered through the blood-brain barrier and drastically inhibited tumor growth in both allograft and orthotopic models of Hh-dependent MB. Our findings reveal that mPEG5kDa-cholane/GlaB is a good candidate for the treatment of Hh-driven tumors and provide relevant implications for the translation of GlaB into clinical practice.

Targeting Pro-Tumoral Macrophages in Early Primary and Metastatic Breast Tumors with the CD206-Binding mUNO Peptide.

M2-like tumor-associated macrophages (M2 TAMs) play important roles in the resistance of tumors to immunotherapies. Selective depletion or reprogramming of M2 TAMs may sensitize the nonresponsive tumors for immune-mediated eradication. However, precision delivery of payloads to M2 TAMs, while sparing healthy tissues, has remained an unresolved challenge. Here, we studied the application of a short linear peptide (CSPGAK, "mUNO") for the delivery of molecular and nanoscale cargoes in M2 TAMs in vitro and the relevance of the peptide for in vivo targeting of early-stage primary breast tumors and metastatic lung foci. First, we performed in silico modeling and found that mUNO interacts with mouse CD206 via a binding site between lectin domains CTLD1 and CTLD2, the same site previously demonstrated to be involved in mUNO binding to human CD206. Second, we showed that cultured M2 macrophages take up fluorescein-labeled (FAM) polymersomes conjugated with mUNO using the sulfhydryl group of its N-terminal cysteine. Pulse/chase studies of FAM-mUNO in M2 macrophages suggested that the peptide avoided lysosomal entrapment and escaped from early endosomes. Third, our in vivo studies with FAM-mUNO demonstrated that intraperitoneal administration results in better pharmacokinetics and higher blood bioavailability than can be achieved with intravenous administration. Intraperitoneal FAM-mUNO, but not FAM-control, showed a robust accumulation in M2-skewed macrophages in mouse models of early primary breast tumor and lung metastasis. This targeting was specific, as no uptake was observed in nonmalignant control organs, including the liver, or other cell types in the tumor, including M1 macrophages. Collectively, our studies support the application of the CD206-binding mUNO peptide for delivery of molecular and nanoscale cargoes to M2 macrophages and manifest the relevance of this mode of targeting primary and metastatic breast tumors.