Synthetic Nanoparticles for Vaccines and Immunotherapy

The immune system plays a critical role in our health. No other component of human physiology plays a decisive role in as diverse an array of maladies, from deadly diseases with which we are all familiar to equally terrible esoteric conditions: HIV, malaria, pneumococcal and influenza infections; cancer; atherosclerosis; autoimmune diseases such as lupus, diabetes, and multiple sclerosis. The importance of understanding the function of the immune system and learning how to modulate immunity to protect against or treat disease thus cannot be overstated. Fortunately, we are entering an exciting era where the science of immunology is defining pathways for the rational manipulation of the immune system at the cellular and molecular level, and this understanding is leading to dramatic advances in the clinic that are transforming the future of medicine.1,2 These initial advances are being made primarily through biologic drugs– recombinant proteins (especially antibodies) or patient-derived cell therapies– but exciting data from preclinical studies suggest that a marriage of approaches based in biotechnology with the materials science and chemistry of nanomaterials, especially nanoparticles, could enable more effective and safer immune engineering strategies. This review will examine these nanoparticle-based strategies to immune modulation in detail, and discuss the promise and outstanding challenges facing the field of immune engineering from a chemical biology/materials engineering perspectiveNational Institutes of Health (U.S.) (Grants AI111860, CA174795, CA172164, AI091693, and AI095109)United States. Department of Defense (W911NF-13-D-0001 and Awards W911NF-07-D-0004

Quantitative single-molecule imaging of TLR4 reveals ligand-specific receptor dimerization

In humans, invading pathogens are recognized by Toll-like receptors (TLRs). Upon recognition of lipopolysaccharide (LPS) derived from the cell wall of gram-negative bacteria, TLR4 dimerizes and can stimulate two different signaling pathways, the proinflammatory, MyD88-dependent pathway and the antiviral, MyD88-independent pathway. The balance between these two pathways is ligand-dependent, and ligand composition determines whether the invading pathogen activates or evades the host immune response. We investigated the dimerization behavior of TLR4 in intact cells in response to different LPS chemotypes through quantitative single-molecule localization microscopy (SMLM). Quantitative super-resolved data showed that TLR4 was monomeric in the absence of its coreceptors MD2 and CD14 in transfected HEK 293 cells. When TLR4 was present together with MD2 and CD14, but in the absence of LPS, 52% of the receptors were monomeric and 48% were dimeric. LPS from Escherichia coli or Salmonella minnesota caused the formation of dimeric TLR4 complexes, whereas the antagonistic LPS chemotype from Rhodobacter sphaeroides maintained TLR4 in monomeric form at the cell surface. Furthermore, we showed that LPS-dependent dimerization was required for the activation of NF-κB signaling. Together, these data demonstrate ligand-dependent dimerization of TLR4 in the cellular environment, which could pave the way for a molecular understanding of biased signaling downstream of the receptor

Nanomedicines for cell reprogramming in tissue engineering and immunomodulation

The main goal of this thesis has been the development of various RNA-based therapies to modulate cell phenotypes in two different disease contexts: cancer and tissue engineering. Given the poor cell-permeability of naked RNA molecules, as well as their short half-life in biorelevant media, a critical aspect of the work has been the design and development of biomaterial-based systems intended to overcome these limitations. On the one hand, we developed a nanocarrier capable of co-allocating RNAi and chemokines to revert the tumor immunosuppression mediated by myeloid derived suppressor cells. On the other hand, we investigated a new concept of gene-activated matrices (GAMs) activated with mRNA-encoded transcription factors (TFs) to direct stem cell chondrogenic and myogenic specification. We finally optimized GAM mechanics in order to enhance TF transfection and cell differentiation driven by TF overexpression

Therapeutic targeting in the silent era: advances in non-viral siRNA delivery

Gene silencing using RNA-interference, first described in mammalian systems almost a decade ago, is revolutionizing therapeutic target validation efforts both in vitro and in vivo. Moreover, the potential for using short interfering RNA (siRNA) as a therapy in its own right is also progressing at a significant pace. However, the widespread use of such approaches is contingent on having appropriate systems to achieve clinically appropriate, safe, and efficient delivery of siRNA. There are many physicochemical and biological barriers to such delivery, and a growing emphasis on the design and characterisation of non-viral technologies that will overcome these barriers and expedite targeted delivery. This review discusses the considerations and challenges associated with use of siRNA-based therapeutics, including stability and off-target effects. Speculation is made on the properties of an ideal delivery system and the non-viral delivery approaches used to date, both in vitro and in vivo, are classified and discussed. Moreover, the ability of cyclodextrin-based delivery vectors to fulfil many of the criteria of an ideal delivery construct is also elaborated

Nanotechnology for the modulation of the immune response in HIV and cancer

Nanotechnologies with the ability to modulate the immune system can be exploited to develop new advanced therapies. The first part of this thesis describes a potential HIV vaccine candidate consisting of nanoparticles loaded with up to three peptide antigens. Based on the good responses in terms of protection against viral infection in macaques, the quality-by-design and scaling-up production of this vaccine candidate was performed. Finally, in the context of cancer, a potential immunotherapy with the capacity to polarize tumor-associated macrophages towards anti-tumoral phenotypes was developed

Emerging Cationic Nanovaccines

Cationic vaccines of nanometric sizes can directly perform the delivery of antigen(s) and immunomodulator(s) to dendritic cells in the lymph nodes. The positively charged nanovaccines are taken up by antigen-presenting cells (APCs) of the lymphatic system often originating the cellular immunological defense required to fight intracellular microbial infections and the proliferation of cancers. Cationic molecules imparting the positive charges to nanovaccines exhibit a dose-dependent toxicity which needs to be systematically addressed. Against the coronavirus, mRNA cationic nanovaccines evolved rapidly. Nowadays cationic nanovaccines have been formulated against several infections with the advantage of cationic compounds granting protection of nucleic acids in vivo against biodegradation by nucleases. Up to the threshold concentration of cationic molecules for nanovaccine delivery, cationic nanovaccines perform well eliciting the desired Th 1 improved immune response in the absence of cytotoxicity. A second strategy in the literature involves dilution of cationic components in biocompatible polymeric matrixes. Polymeric nanoparticles incorporating cationic molecules at reduced concentrations for the cationic component often result in an absence of toxic effects. The progress in vaccinology against cancer involves in situ designs for cationic nanovaccines. The lysis of transformed cancer cells releases several tumoral antigens, which in the presence of cationic nanoadjuvants can be systemically presented for the prevention of metastatic cancer. In addition, these local cationic nanovaccines allow immunotherapeutic tumor treatment

Targeting immunosuppressive myeloid cells using nanocarriers to improve cancer immunotherapy

In the last decade, different cancer immunotherapy approaches have been proved to produce objective clinical responses and survival benefits in cancer patients who failed conventional treatment options. However, the efficacy of cancer immunotherapy is known to be limited by tumor-induced expansion of immunosuppressive and tumor-promoting immune populations, including cells of myeloid origin (such as tumor associated macrophages –TAMs-, and myeloid-derived suppressor cells –MDSCs-). The development of increasingly powerful and widely applicable immunotherapies is therefore dependent on the availability of supporting treatments able to reduce tumor-associated immunosuppression, with good efficacy and low toxicity. Drug delivery nanosystems have been shown to improve pharmacological proprieties of anti-cancer drugs by increasing drug half-life, enhancing accumulation into tumor tissues and reducing off-target toxicity. In addition, the use of RNA delivery nanosystems is currently being explored for the development of RNA intereference (RNAi)-based anti-cancer therapeutics.The use of drug and RNA delivery nanosystems to target tumor-associated immune cells, besides tumor cells, is a far less explored approach, which is currently showing promiseas tool to improve cancer immunotherapy. In the present work we investigated two different nanotechnology-based tools to modulate the presence and function of tumor-associated myeloid cells:a modified form of gemcitabine encapsulated into lipid nanocapsules (LNC-GemC12), and ployarginine-coated nanocapsules (PolyArgNCs) loaded with short hairpin (sh) RNAs, for in vivo RNAi-based gene silencing. LNC-GemC12 and free gemcitabine hydrochloride (GemHCl, the current standard gemcitabine formulation)were found to selectively deplete both splenic and tumor-infiltrating monocytic (M-) MDSCs, following administration at a very low drug dose to EG7-OVA tumor bearing mice. Remarkably, LNC-GemC12 administration was associated with a stronger and more durable reduction of tumor-infiltrating M-MDSCs as compared withGemHCl, which resulted in the attenuation of MDSC suppressive activity towards T cells. More importantly,treatment of EG7-OVA tumor bearing mice with LNC-GemC12,prior to adoptive cell transfer therapy (ACT) with OVA-specific T lymphocytes,significantly extended mouse survival as compared to mice receiving ACT alone. Conversely, preconditioning with GemHCl at the same dose did not result in a similar survival benefit. PolyArg NCs were loaded with a fluorinated shRNA specific for mouse C/EBPβ transcription factor, which is known to be required for tumor-induced MDSC expansion and acquisition of immunosuppressive functions.PolyArgNCs loaded with the C/EBPβ-specific shRNA (NC-shC/EBPβ) efficiently down-regulated target gene expression in an immortalized MDSC cell line.In vivo, we reported a significant reduction of C/EBPβ mRNA levels in splenic and tumor-infiltrating myeloid cells, following repeated administration of NC-shC/EBPβ to mice bearing MCA203 subcutaneous sarcomas. The present datasupport the use of LNC-GemC12 as MDSC-targeted agent in cancer immunotherapy, notably in combination with ACT. As compared with standard MDSC-depleting chemotherapeutic drug formulations, LNC-GemC12 bears the potential of achieving significant effects at very low, likely not toxic, drug doses. Moreover, the use of a drug already employed in clinical oncology, combined with a biocompatible delivery nanosystem, might facilitate clinical translation.We also provided an initial evidence that shRNA-loaded PolyArg NCs allow to downregulate target gene expression in myeloid cellsin vivo, and could be exploited to therapeutically modulate tumor-associated myeloid cells.

Active-targeted Nanotherapy as Smart Cancer Treatment

Drug delivery systems (DDS) can be designed to improve the pharmacological and therapeutic properties of drugs. Targeted drug delivery, sometimes called smart drug delivery, is a method of delivering medication to a patient in a manner that increases the concentration of the medication in infective organs or cells, relative to others. Cancer is one of the major causes of mortality worldwide and innovative methods for cancer therapy are urgently required. Nanoparticles (NPs), by using active targeting strategy, can enhance the intracellular concentration of drugs in cancerous cells while avoiding toxicity in normal cells. Nanoparticles with bioscience are being actively developed for in vivo tumor imaging, bimolecular profiling of cancer biomarkers, and targeted drug delivery. The advantages of the targeted release system are the reduction in the frequency of dosages taken by the patient, having a uniform effect of the drug, reduction of drug side effects, and reduced fluctuation in circulating drug levels. In this chapter, we focus on targeted drug delivery systems integrated from nanobiotechnology

Nanomedical research and development in Spain: improving the treatment of diseases from the nanoscale

49 p.-7 fig.-9 tab.The new and unique possibilities that nanomaterials offer have greatly impacted biomedicine, from the treatment and diagnosis of diseases, to the specific and optimized delivery of therapeutic agents. Technological advances in the synthesis, characterization, standardization, and therapeutic performance of nanoparticles have enabled the approval of several nanomedicines and novel applications. Discoveries continue to rise exponentially in all disease areas, from cancer to neurodegenerative diseases. In Spain, there is a substantial net of researchers involved in the development of nanodiagnostics and nanomedicines. In this review, we summarize the state of the art of nanotechnology, focusing on nanoparticles, for the treatment of diseases in Spain (2017–2022), and give a perspective on the future trends and direction that nanomedicine research is taking.This work has been partially supported by MCIN/AEI /10.13039/501100011033 and European Union NextGenerationEU/PRTR (PID2021-128340OA-I00, PID2020-119352RB-I00, PID2021-127033OB-C21 and RTI2018-101050-J-I00), Comunidad de Madrid (S2022/BMD-7403 RENIM-CM and Talento program 2018-T1/IND-1005), European Union’s Horizon 2020 research and innovation programme (grant agreement No 685795), la Caixa Foundation LCF/PR/HA21/52350003, Asociación Española Contra el Cáncer IDEAS21989THOM, MCIN/AEI/10.13039/501100011033 and “ESF investing in your future” (RYC2019-027489-I, RYC2020-029282-I). CTB thanks Ministerio de Educación (FPU18/06310) for the predoctoral fellowship. IMDEA Nanociencia acknowledges support from the ‘Severo Ochoa’ Programme for Centers of Excellence in R&D (MINECO, CEX2020-001039-S).Peer reviewe