Polysaccharide-based self-assembling nanohydrogels: An overview on 25-years research on pullulan

The aim of this overview is to review the evolution of the studies carried out, during more than 25 years, on nanohydrogels obtained by self-assembling of pullulan (PUL) using several hydrophobization strategies. After the first publications, mainly devoted to the preparation and characterization of PUL nanogels, a remarkable number of studies demonstrated how wide can be the field of applications within the main topic of biopharmaceutics. Numerous hydrophilic and lipophilic drugs were entrapped in the nanogel networks, consequently PUL nanogels have been proposed as delivery systems for single drugs and for combination therapies which allowed improvements of pharmacological activities and patient compliance. Furthermore, the large amount of water content allowed loading also proteins which could maintain their native structure and properties. Stimuli-sensitive and stealth PUL nanogel formulations allowed improving the performances of antitumor drugs. These nanohydrogels have also been studied for imaging techniques and for vaccines to be administered by injection and by mucosal application. The studies on PUL nanogels are still in progress and the perspectives for future researches are also addressed

Anti-HIV therapy: pipeline approaches and future directions

Human immunodeficiency virus (HIV), with about 30 million deaths and double infections (in developing countries), is an open challenge today for global scientists. Developing safe and effective measurements against it has become the prime need of hour. Though, putting it at health priority, various efforts like chemotherapy, vaccines and others are attempted globally over last decade. Consequently, highly active antiretroviral therapy was introduced but fails to completely block the viral replication due to drug resistance and various other severe side effects. The antigenic variability and lack of appropriate experimental models is the major obstacle in the development of an ever effective treatment against HIV. However, to overcome the present hurdles and to emerge a preventive HIV vaccine efforts at various platforms are done. A renewed, coordinated research, preclinical studies, clinical trials together with sufficient long term scientific and commercial commitments are made. Few of the therapeutic efforts viz. RNA interference (RNAi) based replication arrest of HIV, viral enzymes’ inhibitors, nanotechnology based HIV control and various preclinically trialed vaccines are reviewed in this paper. Also, the observed toxicity of existing therapeutic regimen, key challenges and future prospects for the development of better tolerated prophylactic HIV-1 vaccine are discussed

Nanoparticles in the treatment of chronic lung diseases

Nanoparticles, although considered a topic of modern medicine, actually have an interesting history. Currently, advances in nanomedicine hold great promise as drug carrier systems for sustained release and targeted delivery of diverse therapeutic agents. Nanoparticles can be defined as complex drug carrier systems which incorporate and protect a certain drug or particle. Nanoparticles can be administered via different routes, such as intravenous injection, oral administration, or pulmonary inhalation. Even though the use of nano-carriers via pulmonary inhalation is heavily debated, this system represents an attractive alternative to the intravenous or oral routes, due to the unique anatomical and physiological features of the lungs and the minimal interactions between the targeted site and other organs. Some of the widely used nano-carriers for the treatment of chronic pulmonary diseases, via pulmonary route, are as follows: polymeric nanoparticles, liposomal nano-carriers, solid lipid nanoparticles, and submicron emulsions. Nano-carrier systems provide the advantage of sustained-drug release in the lung tissue resulting in reduced dosing frequency and improved patient compliance. Further studies focusing on understanding the mechanisms of action of nanoparticles and improving their chemical structure are required in order to better understand the potential long-term risk of excipient toxicity and nanoscale carriers

Self-assembled hydrogel nanoparticles for drug delivery applications

Hydrogel nanoparticles—also referred to as polymeric nanogels or macromolecular micelles—are emerging as promising drug carriers for therapeutic applications. These nanostructures hold versatility and properties suitable for the delivery of bioactive molecules, namely of biopharmaceuticals. This article reviews the latest developments in the use of self-assembled polymeric nanogels for drug delivery applications, including small molecular weight drugs, proteins, peptides, oligosaccharides, vaccines and nucleic acids. The materials and techniques used in the development of self-assembling nanogels are also described

Anti-tumor immunity induced by a novel nanoparticulate vaccine : a mechanistic approach

Tese de doutoramento, Farmácia (Tecnologia Farmacêutica), Universidade de Lisboa, Faculdade de Farmácia, 2016Biodegradable polymeric nanoparticles (NP) are promising tools for tumor eradication. Different nanovaccines based on the aliphatic-polyester (poly(lactic-co-glycolic acid) (PLGA) have been developed and optimized to investigate how different methods for antigen association to nanoparticulate carriers affect antigen uptake by antigen presenting cells (APC) and the generation and nature of antigen-specific immune responses. The different experimental parameters have been tested in order to predict the effect of the nature of protein association (adsorbed vs. entrapped) and polymers/surfactant concentrations, on NP average mean diameter, polydispersity index, surface charge, encapsulation efficiency, protein integrity and surfactant residual amount. This development procedure allowed for the rational identification of particle composition and experimental conditions that led to the antigenassociated nanocarrier presenting the ideal product specifications previously identified for optimal immune cell modulation, having in consideration literature evidences and our previous data. We used PLGA and pegylated (PEG)-PLGA as a matrix polymer. When preparing NP with double-emulsion solvent-evaporation technique, we used α-lactalbumin (LALBA) as an antigen, glycol chitosan (CS) to increase the viscosity of the internal aqueous phase (IP) and polyvinyl alcohol (PVA) or Pluronic F127 (PF127) to stabilization the interphase of double emulsion. Various concentrations of surfactants has been used (2, 4, 5, 8, 10, 12 % v/v) to best meet the criteria of the most optimal nanoparticulate delivery system. We aimed for NP below 200 nm, polydispersity index (PDI) under 0.2 and ζ potential close to neutrality. The formulations with 10 % v/v PVA solution in IP and 0.3 % v/v PVA or PF127 in external aqueous phase (EP) presented stable and repetitive NP formulations with desired physiochemical properties. The highest encapsulation efficacy (EE) values were obtained for the formulation prepared with PVA (EntrapLALBA(PVA)), reaching 80 % of the integrated protein, while NP with PF127 (EntrapLALBA(PF127)) presented around 70 % EE. As regards the protein adsorbed, NP formulations prepared with PVA (AdsLALBA(PVA)) or PF127 (AdsLALBA(PF127), reached the values of 40 % and 50 %, respectively. Dynamic light scattering (DLS) and laser doppler velocimetry (LDV) were used to determine average particle size, polydispersity (PDI) and ζ potential, respectively. EntrapLALBA(PVA) presented the average size of 171 nm with PDI around 0.15 and ζ potential 0.51 mV. EntrapLALBA(PF127) had 185 nm with PDI 0.17 and ζ potential 0.37 mV. On the other side AdsLALBA(PVA) and AdsLALBA(PF127) presented slightly biggersize 188 nm and 195 nm, respectively, with more negative ζ potential, -1,89 and -1,42 mV, respectively. NP size, PDI and geometry were also confirmed by atomic force microscopy (AFM). Protein integrity was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Fourier transform infrared spectroscopy (FTIR) was further used to study the interaction of protein adsorption onto surfaces of polymeric NP. The presence of bands at the wavelengths 1662 cm-1 and 1562 cm-1, that are specific for primary amides and amines, were detected in formulations AdsLALBA(PVA) and AdsLALBA(PF127), where LALBA was adsorbed onto the surface of the NP. While in NP EntrapLALBA(PVA) and EntrapLALBA(PF127) this bands were absent indicating the protein incorporation into the polymeric matrix. Differential scanning calorimetry (DSC) showed that the formulation process did not alter the structure of the polymers used in the preparation of NP. The ability of the optimal formulations for systemic delivery and activation of DC was further evaluated in vitro and in vivo on bone marrow derived dendritic cells (BMDC). Similar activation and maturation profile of APC were obtained after the internalization of NP with antigen entrapped (EntrapNP) or adsorbed onto NP surface (AdsNP) evaluated at 3, 6, 16 and 24 h (ImageStreamTM and flow cytometry). The DC-NP interaction increased with the incubation time, presenting internalization values between 50-60% and 30-40%, in vitro and in vivo, respectively. Interestingly, MHCII was upregulated after the immunization with AdsNP and MHCI after the vaccination with EntrapNP, suggesting more efficient crosspresentation. To evaluate the activation of antigen-specific immune response, we replaced LALBA for ovalbumin (OVA). It is a highly immunogenic model antigen for which, in immunology, we have well-established animal models that can help to study the effect of the antigen on immune system without any unspecific immune response. Phenotype, frequency and efficacy of immune cell stimulation induced by NP loaded with OVA (EntrapNP or AdsNP) and with/without TLR ligands (CpG and Monophosphoryl Lipid A (MPLA)) were characterized and their specificity clarified by the engrafted OT-I (CD8+) and OT-II (CD4+) T cells. EntrapNP with OVA and adjuvants presented the strongest antigen-specific cytotoxic immune response, following NP with AdsOVA-Ajds. Moreover, long-lasting memory of cytotoxic T lymphocytes (CTL), was evaluated 8 weeks after a single immunization in response to the different ways of antigen delivery, PBS, OVA in solution, OVA & Adjs in solution and NP AdsOVA, AdsOVA-Adjs, EntrapOVA, or EntrapOVA-Adjs. Immunizations where we used antigen alone did not induce efficient CTL reactivation. Overall, Entrap OVA-Adj NP presented the most robust immune response in inducing the memory, followed by AdsOVAAdjs NP and OVA & Adjs in solution. To explore whether the immunization with formulated NP results in efficient crosspriming, an in vivo killing assay in steady-state conditions was performed. 24 h prior to immunization, animals were engrafted with CD8+ (OT-I) cells. 5 days p.i., mice were injected with an equal mix of targeted and untargeted CD45.1 splenocytes. Targeted cells were pulsed with OVA peptide, SIINFEKL, and labeled with higher dose of CFSE (CFSEhi) while untargeted control cells were labeled with a lower dose of CFSE (CSFElow). The in vivo clearance of both type of grafted cells was evaluated 16 h later in LN and spleens by flow cytometry. All NPs, with or without adjuvants, generated the most efficient in vivo killing activity towards the SIINFEKL-pulsed cells, as shown by the disappearance of the CFSEhi peak of cells. Indicating the successful induction of cross-priming and activation of antigenspecific CTL that could potentially lead to a broad and effective immune response pivotal in case of tumor rejections. To test the efficacy of the therapeutic vaccine and to have a good read-out of the antigen-specific T cells, we used B16.MO5 melanoma-bearing mice using different vaccination schedules, single and three time immunization with OVA and adjuvants- loaded NP. The groups immunized with PBS and Empty NP served as a control and presented similar tumor growth. All treated groups showed a significant reduction in tumor growth. EntrapOVA-Adjs NP (3x immunization) presented the slowest tumor growth (more than 12x smaller final tumor volume), followed by the group immunized with AdsOVA-Adjs NP (3x immunization) (more than 9x smaller final tumor volume) and single vaccination with EntrapOVA-Adjs NP (more than 8x smaller final tumor volume). The highest amount of tumor infiltrating lymphocytes (TILs) at the tumor site was detected for EntrapOVA-Adjs NP (3x immunization), suggesting the best remission and survival prognosis of the treated group. Two inflammatory cytokines (TNF-α and IFN-γ) produced by TILs were quantified in tumor microenvironment. 4-time high levels of TNF-α upon 3-time immunizations were detected in tumor microenvironment which kept the tumor growth under control. On the other side in the spleens, repeated vaccination prevented upregulation of both inflammatory markers, TNF-α and IFN-γ, indicating absence of systemic inflammation. Overall, the present work shows that the design of safe but efficacious nanoparticulate cancer vaccines requires a deep understanding of NP biological effect on immune cells, both under steady-state and cancerous environment. A detailed characterization of the imune cell-related pathways modulated by the optimal antigen-loaded NP and their correlation with the nature and anti-tumor efficacy of the induced immune response was performed in vivo in healthy and pathological conditions, showing that the nature of antigen delivery is crucial for a specific T cell activation and targeted cytotoxic effect

Biomaterial Strategies for Immunomodulation

Strategies to enhance, suppress, or qualitatively shape the immune response are of importance for diverse biomedical applications, such as the development of new vaccines, treatments for autoimmune diseases and allergies, strategies for regenerative medicine, and immunotherapies for cancer. However, the intricate cellular and molecular signals regulating the immune system are major hurdles to predictably manipulating the immune response and developing safe and effective therapies. To meet this challenge, biomaterials are being developed that control how, where, and when immune cells are stimulated in vivo, and that can finely control their differentiation in vitro. We review recent advances in the field of biomaterials for immunomodulation, focusing particularly on designing biomaterials to provide controlled immunostimulation, targeting drugs and vaccines to lymphoid organs, and serving as scaffolds to organize immune cells and emulate lymphoid tissues. These ongoing efforts highlight the many ways in which biomaterials can be brought to bear to engineer the immune system.Bill & Melinda Gates FoundationUnited States. Army Research Office. Institute for Soldier Nanotechnologies (Contract W911NF-13-D-0001)Ragon Institute of MGH, MIT and HarvardCancer Research Institute (New York, N.Y.) (Irvington Postdoctoral Fellowship)National Institutes of Health (U.S.) (Awards AI104715, CA172164, CA174795, and AI095109

Polymeric nanogels as vaccine delivery systems

Polymeric nanogels find a relevant field of application in the formulation of a new generation of therapeutic and preventive vaccines, aiming at the fine-tuned modulation of the immune response. Intrinsic properties of polymeric nanogels, such as material chemistry, size and shape, surface charge, and hydrophobicity or hydrophilicity, may be determining factors in shaping the induced immune response. These materials can thus work as synthetic adjuvants, which can also be conjugated with immunostimulants. Polymeric nanogels protect vaccine antigens from degradation in vivo and, surface-conjugated with antibodies or specific ligands, could increase active targeting specificity. This review covers the recent published data concerning the modulation of innate and adaptive immune responses by engineered polymeric nanogels and their potential application as delivery systems in vaccination.S.A. Ferreira is the recipient of a fellowship from International Iberian Nanotechnology Laboratory (INL)

Nanotechnology Synergised Immunoengineering for Cancer

Novel strategies modulating the immune system yielded enhanced anticancer responses and improved cancer survival. Nevertheless, the success rate of immunotherapy in cancer treatment has been below expectation(s) due to unpredictable efficacy and off-target effects from systemic dosing of immunotherapeutic. As a result, there is an unmet clinical need for improving conventional immunotherapy. Nanotechnology offers several new strategies, multimodality, and multiplex biological targeting advantage to overcome many of these challenges. These efforts enable programming the pharmacodynamics, pharmacokinetics, delivery of immunomodulatory agents/co-delivery of compounds to prime at the tumor sites for improved therapeutic benefits. This review provides an overview of the design and clinical principles of biomaterials driven nanotechnology and their potential use in personalized nanomedicines, vaccines, localized tumor modulation, and delivery strategies for cancer immunotherapy. In this review, we also summarize the latest highlights and recent advances in combinatorial therapies avail in the treatment of cold and complicated tumors. It also presents key steps and parameters implemented for clinical success. Finally, we analyse, discuss, and provide clinical perspectives on the integrated opportunities of nanotechnology and immunology to achieve synergistic and durable responses in cancer treatment

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