3D Printing as a Strategy to Scale-Up Biohybrid Hydrogels for T Cell Manufacture.

The emergence of cellular immunotherapy treatments is introducing more efficient strategies to combat cancer as well as autoimmune and infectious diseases. However, the cellular manufacturing procedures associated with these therapies remain costly and time-consuming, thus limiting their applicability. Recently, lymph-node-inspired PEG-heparin hydrogels have been demonstrated to improve primary human T cell culture at the laboratory scale. To go one step further in their clinical applicability, we assessed their scalability, which was successfully achieved by 3D printing. Thus, we were able to improve primary human T cell infiltration in the biohybrid PEG-heparin hydrogels, as well as increase nutrient, waste, and gas transport, resulting in higher primary human T cell proliferation rates while maintaining the phenotype. Thus, we moved one step further toward meeting the requirements needed to improve the manufacture of the cellular products used in cellular immunotherapies.

Three-dimensional cell culture of chimeric antigen receptor T cells originated from peripheral blood mononuclear cells towards cellular therapies.

BACKGROUND AIMS: With the objective of improving the ex vivo production of therapeutic chimeric antigen receptor (CAR) T cells, we explored the addition of three-dimensional (3D) polystyrene scaffolds to standard suspension cell cultures. METHODS: We aimed to mimic the structural support given by the lymph nodes during in vivo lymphocyte expansion. RESULTS: We observed an increase in cell proliferation compared with standard suspension systems as well as an enhanced cytotoxicity toward cancer cells. Moreover, we directly obtained the CAR T cells from peripheral blood mononuclear cells, thus minimizing the ex vivo manipulation of the therapeutic cells and opening the way to synergies among different cell populations. CONCLUSIONS: We propose the use of commercially available 3D polystyrene systems to improve the current immune cell cultures and resulting cell products for emerging cellular (immuno)therapies.

Enhanced human T cell expansion with inverse opal hydrogels.

Advanced personalized immunotherapies still have to overcome several biomedical and technical limitations before they become a routine cancer treatment in spite of recent achievements. In adoptive cell therapy (ACT), the capacity to obtain adequate numbers of therapeutic T cells in the patients following ex vivo treatment should be improved. Moreover, the time and costs to produce these T cells should be reduced. In this work, inverse opal (IOPAL) 3D hydrogels consisting of poly(ethylene) glycol (PEG) covalently combined with heparin were engineered to resemble the environment of lymph nodes, where T cells get activated and proliferate. The introduction of an IOPAL strategy allowed a precise control on the porosity of the hydrogels, providing an increase in the proliferation of primary human CD4+ T cells, when compared with state-of-the-art expansion systems. Additionally, the IOPAL hydrogels also showed a superior expansion compared to hydrogels with the same composition, but without the predetermined pore structure. In summary, we have shown the beneficial effect of having an IOPAL architecture in our 3D hydrogels to help achieving large numbers of cells, while maintaining the desired selected phenotypes required for ACT.

Methods for the Characterization of Protein Aggregates.

The physicochemical characterization of protein aggregates yields an important contribution to further our understanding on many diseases for which the formation of protein aggregates is one of the pathological hallmarks. On the other hand, bacterial inclusion bodies (IBs) have recently been shown to be highly pure proteinaceous aggregates of a few hundred nanometers, produced by recombinant bacteria supporting the biological activities of the embedded polypeptides. Despite the wide spectrum of uses of IBs as functional and biocompatible materials upon convenient engineering, very few is known about their physicochemical properties.In this chapter we present methods for the characterization of protein aggregates as particulate materials relevant to their physicochemical and nanoscale properties.Specifically, we describe the use of dynamic light scattering (DLS) for sizing, nanoparticle tracking analysis for sizing and counting, and zeta potential measurements for the determination of colloidal stability. To study the morphology of protein aggregates we present the use of atomic force microscopy (AFM) and scanning electron microscopy (SEM). Cryo-transmission electron microscopy (cryo-TEM) will be used for the determination of the internal structuration. Moreover, wettability and nanomechanical characterization can be performed using contact angle (CA) and force spectroscopic AFM (FS-AFM) measurements of the proteinaceous nanoparticles, respectively. Finally, the 4'4-dithiodipyridine (DTDP) method is presented as a way of relatively quantifying accessible sulfhydryl groups in the structure of the nanoparticle .The physical principles of the methods are briefly described and examples are given to help clarify capabilities of each technique.

Engineering pH-Sensitive Stable Nanovesicles for Delivery of MicroRNA Therapeutics.

MicroRNAs (miRNAs) are small non-coding endogenous RNAs, which are attracting a growing interest as therapeutic molecules due to their central role in major diseases. However, the transformation of these biomolecules into drugs is limited due to their unstability in the bloodstream, caused by nucleases abundantly present in the blood, and poor capacity to enter cells. The conjugation of miRNAs to nanoparticles (NPs) could be an effective strategy for their clinical delivery. Herein, the engineering of non-liposomal lipid nanovesicles, named quatsomes (QS), for the delivery of miRNAs and other small RNAs into the cytosol of tumor cells, triggering a tumor-suppressive response is reported. The engineered pH-sensitive nanovesicles have controlled structure (unilamellar), size (<150 nm) and composition. These nanovesicles are colloidal stable (>24 weeks), and are prepared by a green, GMP compliant, and scalable one-step procedure, which are all unavoidable requirements for the arrival to the clinical practice of NP based miRNA therapeutics. Furthermore, QS protect miRNAs from RNAses and when injected intravenously, deliver them into liver, lung, and neuroblastoma xenografts tumors. These stable nanovesicles with tunable pH sensitiveness constitute an attractive platform for the efficient delivery of miRNAs and other small RNAs with therapeutic activity and their exploitation in the clinics.

Impact of Chemical Composition on the Nanostructure and Biological Activity of α-Galactosidase-Loaded Nanovesicles for Fabry Disease Treatment.

Fabry disease is a rare lysosomal storage disorder characterized by a deficiency of α-galactosidase A (GLA), a lysosomal hydrolase. The enzyme replacement therapy administering naked GLA shows several drawbacks including poor biodistribution, limited efficacy, and relatively high immunogenicity in Fabry patients. An attractive strategy to overcome these problems is the use of nanocarriers for encapsulating the enzyme. Nanoliposomes functionalized with RGD peptide have already emerged as a good platform to protect and deliver GLA to endothelial cells. However, low colloidal stability and limited enzyme entrapment efficiency could hinder the further pharmaceutical development and the clinical translation of these nanoformulations. Herein, the incorporation of the cationic miristalkonium chloride (MKC) surfactant to RGD nanovesicles is explored, comparing two different nanosystems-quatsomes and hybrid liposomes. In both systems, the positive surface charge introduced by MKC promotes electrostatic interactions between the enzyme and the nanovesicles, improving the loading capacity and colloidal stability. The presence of high MKC content in quatsomes practically abolishes GLA enzymatic activity, while low concentrations of the surfactant in hybrid liposomes stabilize the enzyme without compromising its activity. Moreover, hybrid liposomes show improved efficacy in cell cultures and a good in vitro/in vivo safety profile, ensuring their future preclinical and clinical development.

CCL21-loaded 3D hydrogels for T cell expansion and differentiation.

Recent achievements in the field of immunotherapy, such as the development of engineered T cells used in adoptive cell therapy, are introducing more efficient strategies to combat cancer. Nevertheless, there are still many limitations. For example, these T cells are challenging to manufacture, manipulate, and control. Specifically, there are limitations in producing the large amounts of therapeutic T cells needed for these therapies in a short period of time and in an economically viable manner. In this study, three-dimensional (3D) poly(ethylene) glycol (PEG) hydrogels covalently combined with low molecular weight heparin are engineered to resemble the lymph nodes, where T cells reproduce. In these hydrogels, PEG provides the needed structural and mechanical properties, whereas heparin is used as an anchor for the cytokine CCL21, which is present in the lymph nodes, and can affect cell migration and proliferation. The 3D structure of the hydrogel in combination with its loading capacity result in an increased primary human CD4+ T cell proliferation compared to the state-of-the-art expansion systems consisting of artificial antigen presenting cells. Thus, we present a new tool for adoptive cell therapy to help achieving the large numbers of cells required for therapy of selected phenotypes targeted against cancer cells, by mimicking the lymph nodes.

Stable anchoring of bacteria-based protein nanoparticles for surface enhanced cell guidance.

In tissue engineering, biological, physical, and chemical inputs have to be combined to properly mimic cellular environments and successfully build artificial tissues which can be designed to fulfill different biomedical needs such as the shortage of organ donors or the development of in vitro disease models for drug testing. Inclusion body-like protein nanoparticles (pNPs) can simultaneously provide such physical and biochemical stimuli to cells when attached to surfaces. However, this attachment has only been made by physisorption. To provide a stable anchoring, a covalent binding of lactic acid bacteria (LAB) produced pNPs, which lack the innate pyrogenic impurities of Gram-negative bacteria like Escherichia coli, is presented. The reported micropatterns feature a robust nanoscale topography with an unprecedented mechanical stability. In addition, they are denser and more capable of influencing cell morphology and orientation. The increased stability and the absence of pyrogenic impurities represent a step forward towards the translation of this material to a clinical setting.

MKC-Quatsomes: a stable nanovesicle platform for bio-imaging and drug-delivery applications.

Quatsomes are outstanding new lipid-based nanovesicles that are highly homogeneous and stable in different media for years, but the composition must be carefully chosen to avoid any potentially toxic side effects in in vivo applications. To this end, we have developed and studied a novel type of Quatsomes composed of cholesterol and myristalkonium chloride (MKC), the latter being extensively used as antimicrobial preservative in many ophthalmic and parenteral formulations on the EU and USA market. We have synthesized these novel MKC-Quatsomes in different media that are suitable for parenteral administration, and confirmed their stability in these media for 18 months, as well as the stability in human serum for 24 hours. Biodistribution assays were performed after intravenous injection of fluorescently labeled MKC-Quatsomes in live mice bearing xenografted colorectal tumors, showing nanovesicle accumulation in tumors, liver, spleen, and kidneys. No histological alteration or toxicity was observed in any of these organs.

Fully Water-Soluble Polyphosphorhydrazone-Based Radical Dendrimers Functionalized with Tyr-PROXYL Radicals as Metal-Free MRI T1 Contrast Agents.

The finding of alternative imaging probes to Gadolinium (Gd) and other metal based contrast agents (CA) is crucial to overcome their established toxicity. Herein we describe the synthesis and characterization of an entirely organic metal-free magnetic resonance imaging (MRI) contrast agent based on polyphosphorhydrazone (PPH) dendrimers, fully functionalized with up to 48 organic nitroxide radical units. We propose an innovative synthetic procedure based on the use of an amino acid linker (Tyr) coupled to each dendrimer's branch that permits the anchoring of the radicals and at the same time makes possible the control over their water solubility. We demonstrate that the negatively charged resulting PPH Gn-Tyr-PROXYL (n = 0-3) radical dendrimers are excellent candidates to be used as MRI contrast agents, suited for biomedical applications as they show high water solubility, no aggregation problems, and low cytotoxicity, as well as good stability in highly reducing environments. It is achieved a remarkable r1 relaxivity, ca. four times higher (13 mM-1 s-1) than the gold-standard Gd-DTPA used in clinics. Furthermore, the r1 and r2 relaxivity per unit of radical showed an increase with the increase in generation of dendrimers.

Artificial 3D Culture Systems for T Cell Expansion.

Adoptive cell therapy, i.e., the extraction, manipulation, and administration of ex vivo generated autologous T cells to patients, is an emerging alternative to regular procedures in cancer treatment. Nevertheless, these personalized treatments require laborious and expensive laboratory procedures that should be alleviated to enable their incorporation into the clinics. With the objective to improve the ex vivo expansion of large amount of specific T cells, we propose the use of three-dimensional (3D) structures during their activation with artificial antigen-presenting cells, thus resembling the natural environment of the secondary lymphoid organs. Thus, the activation, proliferation, and differentiation of T cells have been analyzed when cultured in the presence of two 3D systems, Matrigel and a 3D polystyrene scaffold, showing an increase in cell proliferation compared to standard suspension systems.

Highly Stable and Red-Emitting Nanovesicles Incorporating Lipophilic Diketopyrrolopyrroles for Cell Imaging.

Diketopyrrolopyrroles (DPPs) have recently attracted much interest as very bright and photostable red-emitting molecules. However, their tendency to form nonfluorescent aggregates in water through the aggregation-caused quenching (ACQ) effect is a major issue that limits their application under the microscope. Herein, two DPP molecules have been incorporated into the membrane of highly stable and water-soluble quatsomes (QS; nanovesicles composed of surfactants and sterols), which allow their nanostructuration in water and, at the same time, limits the ACQ effect. The obtained fluorescent organic nanoparticles showed superior structural homogeneity, along with long-term colloidal and optical stability. A thorough one- (1P) and two-photon (2P) fluorescence characterization revealed the promising photophysical features of these fluorescent nanovesicles, which showed a high 1P and 2P brightness. Finally, the fluorescent QSs were used for the in vitro bioimaging of Saos-2 osteosarcoma cell lines; this demonstrates their potential as nanomaterials for bioimaging applications.

Tuning Spin-Spin Interactions in Radical Dendrimers.

Two generations of polyphosphorhydrazone (PPH) dendrimers were synthesized and fully functionalized with TEMPO radicals via acrylamido or imino group linkers to evaluate the impact of the linker substitution on the radical-radical interactions. A drastic change in the way that the radicals interacted among them was observed by EPR and CV studies: while radicals in Gn -imino-TEMPO dendrimers presented a strong spin-spin interaction, in the Gn -acrylamido-TEMPO ones they acted mainly as independent radicals. This shows that these interactions could be tuned by the solely substitution of the radical linker, opening the perspective of controlling and modulating the extension of these interactions depending on each application. The chemical properties of the linker strongly influence the spin-spin exchange between pendant radicals.

Benznidazole Nanoformulates: A Chance to Improve Therapeutics for Chagas Disease.

This article describes the characterization of various encapsulated formulations of benznidazole, the current first-line drug for the treatment of Chagas disease. Given the adverse effects of benznidazole, safer formulations of this drug have a great interest. In fact, treatment of Chagas disease with benznidazole has to be discontinued in as much as 20% of cases due to side effects. Furthermore, modification of delivery and formulations could have potential effects on the emergence of drug resistance. The trypanocidal activity of new nanostructured formulations of benznidazole to eliminate Trypanosoma cruzi was studied in vitro as well as their toxicity in two cultured mammalian cell lines (HepG2 and Fibroblasts). Nanoparticles tested included nanostructured lipid carriers, solid lipid nanoparticles, liposomes, quatsomes, and cyclodextrins. The in vitro cytotoxicity of cyclodextrins-benznidazole complexes was significantly lower than that of free benznidazole, whereas their trypanocidal activity was not hampered. These results suggest that nanostructured particles may offer improved therapeutics for Chagas disease.

Fluorenyl-Loaded Quatsome Nanostructured Fluorescent Probes.

Delivery of hydrophobic materials in biological systems, for example, contrast agents or drugs, is an obdurate challenge, severely restricting the use of materials with otherwise advantageous properties. The synthesis and characterization of a highly stable and water-soluble nanovesicle, referred to as a quatsome (QS, vesicle prepared from cholesterol and amphiphilic quaternary amines), that allowed the nanostructuration of a nonwater soluble fluorene-based probe are reported. Photophysical properties of fluorenyl-quatsome nanovesicles were investigated via ultraviolet-visible absorption and fluorescence spectroscopy in various solvents. Colloidal stability and morphology of the nanostructured fluorescent probes were studied via cryogenic transmission electronic microscopy, revealing a "patchy" quatsome vascular morphology. As an example of the utility of these fluorescent nanoprobes, examination of cellular distribution was evaluated in HCT 116 (an epithelial colorectal carcinoma cell line) and COS-7 (an African green monkey kidney cell line) cell lines, demonstrating the selective localization of C-QS and M-QS vesicles in lysosomes with high Pearson's colocalization coefficient, where C-QS and M-QS refer to quatsomes prepared with hexadecyltrimethylammonium bromide or tetradecyldimethylbenzylammonium chloride, respectively. Further experiments demonstrated their use in time-dependent lysosomal tracking.

Methods for characterization of protein aggregates.

Physicochemical characterization of protein aggregates is important on one hand, due to its large impact in understanding many diseases for which formation of protein aggregates is one of the pathological hallmarks. On the other hand, recently it has been observed that bacterial inclusion bodies (IBs) are also highly pure proteinaceous aggregates of a few hundred nanometers produced by recombinant bacteria supporting the biological activities of the embedded polypeptides. From this fact arises a wide spectrum of uses of IBs as functional and biocompatible materials upon convenient engineering but very few is known about their physicochemical properties. In this chapter we present methods for the characterization of protein aggregates as particulate materials relevant to their physicochemical and nanoscale properties. Specifically, we describe the use of infrared spectroscopy (IR) for the determination of the secondary structure, dynamic light scattering (DLS) for sizing, nanosight for sizing and counting, and Z-potential measurements for the determination of colloidal stability. To study their morphology we present the use of atomic force microscopy (AFM). Cryo-transmission electron microscopy will be used for the determination of the internal structuration. Moreover, wettability and nanomechanical characterization can be performed using contact angle (CA) and force spectroscopic AFM measurements of the proteinaceous nanoparticles, respectively. The physical principles of the methods are briefly described and examples of data for real samples and how that data is interpreted are given to help clarify capabilities of each technique.

Intracellular targeting of CD44+ cells with self-assembling, protein only nanoparticles.

CD44 is a multifunctional cell surface protein involved in proliferation and differentiation, angiogenesis and signaling. The expression of CD44 is up-regulated in several types of human tumors and particularly in cancer stem cells, representing an appealing target for drug delivery in the treatment of cancer. We have explored here several protein ligands of CD44 for the construction of self-assembling modular proteins designed to bind and internalize target cells. Among five tested ligands, two of them (A5G27 and FNI/II/V) drive the formation of protein-only, ring-shaped nanoparticles of about 14 nm that efficiently bind and penetrate CD44(+) cells by an endosomal route. The potential of these newly designed nanoparticles is evaluated regarding the need of biocompatible nanostructured materials for drug delivery in CD44-linked conditions.

In vivo architectonic stability of fully de novo designed protein-only nanoparticles.

The fully de novo design of protein building blocks for self-assembling as functional nanoparticles is a challenging task in emerging nanomedicines, which urgently demand novel, versatile, and biologically safe vehicles for imaging, drug delivery, and gene therapy. While the use of viruses and virus-like particles is limited by severe constraints, the generation of protein-only nanocarriers is progressively reachable by the engineering of protein-protein interactions, resulting in self-assembling functional building blocks. In particular, end-terminal cationic peptides drive the organization of structurally diverse protein species as regular nanosized oligomers, offering promise in the rational engineering of protein self-assembling. However, the in vivo stability of these constructs, being a critical issue for their medical applicability, needs to be assessed. We have explored here if the cross-molecular contacts between protein monomers, generated by end-terminal cationic peptides and oligohistidine tags, are stable enough for the resulting nanoparticles to overcome biological barriers in assembled form. The analyses of renal clearance and biodistribution of several tagged modular proteins reveal long-term architectonic stability, allowing systemic circulation and tissue targeting in form of nanoparticulate material. This observation fully supports the value of the engineered of protein building blocks addressed to the biofabrication of smart, robust, and multifunctional nanoparticles with medical applicability that mimic structure and functional capabilities of viral capsids.

Hydrophobic gentamicin-loaded nanoparticles are effective against Brucella melitensis infection in mice.

The clinical management of human brucellosis is still challenging and demands in vitro active antibiotics capable of targeting the pathogen-harboring intracellular compartments. A sustained release of the antibiotic at the site of infection would make it possible to reduce the number of required doses and thus the treatment-associated toxicity. In this study, a hydrophobically modified gentamicin, gentamicin-AOT [AOT is bis(2-ethylhexyl) sulfosuccinate sodium salt], was either microstructured or encapsulated in poly(lactic-co-glycolic acid) (PLGA) nanoparticles. The efficacy of the formulations developed was studied both in vitro and in vivo. Gentamicin formulations reduced Brucella infection in experimentally infected THP-1 monocytes (>2-log10 unit reduction) when using clinically relevant concentrations (18 mg/liter). Moreover, in vivo studies demonstrated that gentamicin-AOT-loaded nanoparticles efficiently targeted the drug both to the liver and the spleen and maintained an antibiotic therapeutic concentration for up to 4 days in both organs. This resulted in an improved efficacy of the antibiotic in experimentally infected mice. Thus, while 14 doses of free gentamicin did not alter the course of the infection, only 4 doses of gentamicin-AOT-loaded nanoparticles reduced the splenic infection by 3.23 logs and eliminated it from 50% of the infected mice with no evidence of adverse toxic effects. These results strongly suggest that PLGA nanoparticles containing chemically modified hydrophobic gentamicin may be a promising alternative for the treatment of human brucellosis.

Cellular pharmacokinetics and intracellular activity against Listeria monocytogenes and Staphylococcus aureus of chemically modified and nanoencapsulated gentamicin.

OBJECTIVES: The aim of this study was to investigate different hydrophobic gentamicin formulations [gentamicin-bis(2-ethylhexyl) sulfosuccinate (GEN-AOT), microstructured GEN-AOT (PCA GEN-AOT) and GEN-AOT-loaded poly(lactide-co-glycolide) acid (PLGA) nanoparticles (NPs)] in view of improving its therapeutic index against intracellular bacteria. The intracellular accumulation, subcellular distribution and intracellular activity of GEN-AOT and NPs in different monocytic-macrophagic cell lines were studied. METHODS: Human THP-1 and murine J774 phagocytic cells were incubated with GEN-AOT formulations at relevant extracellular concentrations [from 1× MIC to 18 mg/L (human C(max))], and their intracellular accumulation, subcellular distribution and toxicity were evaluated and compared with those of conventional unmodified gentamicin. Intracellular activity of the formulations was determined against bacteria showing different subcellular localizations, namely Staphylococcus aureus (phagolysosomes) and Listeria monocytogenes (cytosol). RESULTS: GEN-AOT formulations accumulated 2-fold (GEN-AOT) to 8-fold (GEN-AOT NPs) more than gentamicin in phagocytic cells, with a predominant subcellular localization in the soluble fraction (cytosol) and with no significant cellular toxicity. NP formulations allowed gentamicin to exert its intracellular activity after shorter incubation times and/or at lower concentrations. With an extracellular concentration of 10× MIC, a 1 log(10) decrease in S. aureus intracellular inoculum was obtained after 12 h instead of 24 h for NPs versus free gentamicin, and a static effect was observed against L. monocytogenes at 24 h with NPs, while free gentamicin was ineffective. CONCLUSIONS: GEN-AOT formulations yielded a high cellular accumulation, especially in the cytosol, which resulted in improved efficacy against both intracellular S. aureus and L. monocytogenes.