Symposium SM04—Beyond Nano–Challenges and Opportunities in Drug Delivery

While immunotherapy holds great promise for combating cancer, the limited efficacy due to an immunosuppressive tumor microenvironment and systemic toxicity hinder the broader application of cancer immunotherapy. We engineered tumor-targeted lipid-dendrimer-calcium-phosphate (TT-LDCP) nanoparticles (NPs) with thymine-functionalized dendrimers that not only exhibited enhanced gene delivery capacity but also immune adjuvant properties by activating the stimulator of interferon genes (STING)-cGAS pathway. TT-LDCP NPs delivered siRNA against immune checkpoint ligand PD-L1 and immunostimulatory IL-2-encoding plasmid DNA to hepatocellular carcinoma (HCC), increased tumoral infiltration and activation of CD8+ T cells, augmented the efficacy of cancer vaccine immunotherapy, and suppressed HCC progression. Our work presents nanotechnology-enabled dual delivery of siRNA and plasmid DNA that selectively targets and reprograms the immunosuppressive tumor microenvironment to improve cancer immunotherapy.

Introduction: Although responsible for reducing infection, widespread antibiotic use has also created the latest grave challenge in wound care, i.e., antibiotic resistant bacteria. Antibiotic resistance has been making our drug choices for infection control increasingly limited and more expensive. According to the Centers for Disease Control and Prevention (CDC), in the U.S. alone, antibiotic resistant bacteria cause at least 2.8 million infections and 35,000 deaths a year and $55-70 billion per year in economic impact. Meanwhile, it is known that trauma (e.g. open fractures) and major burns lead to decreased resistance to infection (“immunosuppression”) and thereby have high infection rates. Here, we present the engineering of biomaterials with interleukin 12p70 (IL-12) and other cytokines to tune the immune responses locally against open fracture associated infections.
Methods: IL-12 nanocoatings and microcapsules were prepared using electrostatic layer-by-layer (LbL) self-assembly. Polymers like poly(L-lysine) or PLL, poly(L-glutamic acid) or PLGA, and bovine serum albumin (BSA) were used to prepare the coatings, and the release properties and stabilities of the nanocoatings were examined. Next, an open femur fracture infection model was created using Sprague-Dawley rats. The rats’ femurs were fractured, inoculated with 100 mL 10² colony-forming unit (CFU/0.1mL) Staphylococcus aureus, left open for one hour, mimicking the “golden hour” of trauma patients, and then fixed with a stainless steel Kirschner wire (K-wire) with or without IL-12 nanocoatings. Animals were euthanized and samples of blood, bone, and K-wires were collected for analysis.
Results: IL-12 nanocoatings were prepared on orthopedic implant models (e.g., K-wires and stainless steel sheets). The loading and release of IL-12 from polypeptide nanocoatings were tuned and a sustained release of IL-12 for approximately 10 days was achieved by controlling the LbL self-assembly process. Polypeptide microcapsules were also prepared and loaded with IL-12. Our in vivo studies showed that IL-12 nanocoatings and microcapsules reduced open fracture associated infection and were advantageous compared to systemic or percutaneous injections of IL-12.
Discussion and Conclusions: Trauma like open fractures have been reported to result in diminished production of IL-12, reduced type 1 helper T (Th1) responses, and decreased resistance to infection. IL-12 displays multiple biologic effects on immune cells; for instance, IL-12 stimulates natural killer (NK) cells to release interferon γ, induces Th1 cell and cytotoxic T lymphocyte proliferation, and promotes the progression of cell-mediated immunity. Local application of IL-12 may restore the capability of the host’s inherent resistance to infection, and have led to decreased infection in open fractures.
Acknowledgements: This work is supported by the Office of the Assistant Secretary of Defense for Health Affairs under Award No. W81XWH-17-1-0603. We also acknowledge financial support from AO Foundation, Osteosynthesis & Trauma Care Foundation, WVU PSCoR, and WVCTSI.

Photodynamic therapy (PDT), a treatment method using reactive oxygen species (ROS), have recently been widely used in anticancer treatments. In this study, 130nm gold nanoparticles (AuNP) were stepwise coated with high biocompatible polydopamine(PDA) and photosensitizer chlorin-e-6 (Ce6). The PDA on AuNP surface allowed Ce6 to adhere on the particles through amide bond. Herein, we employed of 2 different NIR light wavelength (650nm and 808nm) to achieve PDT effect. Ce6@PDA@AuNP would absorb by 808nm laser light and generate heat by SPR effect, and Ce6 would absorb 650nm laser light and generate ROS, which can cause the death of target cells. To characterize the nanoparticle, DLS, zeta-potential and Raman spectroscopy of Ce6@PDA@AuNP particle were analyzed. There were an increase in hydrodynamic diameter of the AuNP in the order of AuNP (~130nm), PDA@AuNP (~210nm) and Ce6@PDA@AuNP (~240nm). Due to the anionic charge of PDA, the surface charge of the AuNP decreases after PDA coating, and Raman spectroscopy shows that Ce6@PDA@AuNP shows sharper peak at 1480cm^-1 and 1580cm^-1 than those of PDA@AuNP, and the 983cm^-1 new peaks indicating that Ce6 is attached to the nanoparticle. Besides, Ce6 release behaviors under several pHs were recorded. The more Ce6 release was observed in the lower pH with laser irradiation. Moreover, when Ce6@PDA@AuNP were irradiated with lasers (650nm and 808nm), it was confirmed that the temperature would rise to 58°C and the PDA layer surrounding the AuNP was consequently peeled off after 20 minutes of the irradiation resulted in the release of Ce6 and enhanced ROS generation under the irradiation of 650nm laser. Likewise, since PDA affected by pH, more Ce6 was released from nanoparticles at lower pH, even during the same laser irradiation time resulting better cell apoptosis effect in vitro. These results suggested that Ce6@PDA@AuNP has antitumor effects under near-infrared (NIR) irradiation based on the PDT effect.

Cancer is one of the main threats to human health, due to the large number of cases and high mortality rates. These rates are only expected to increase in future years. Although radiotherapy is the main course of treatment, it is not specific for tumor cells, affecting also healthy cells. Radiosensitizers provide a novel and simple solution to this problem, since they increase the amount of radiation a cell can absorb. This allows for cancer treatments that use a lower dose of radiation, leading to less secondary effects for the patient. Our group synthesized bismuth sulfide (Bi₂S₃) nanoscale particles using a hot injection method followed by a ligand exchange, for use as radiosensitizers. We synthetized Bi₂S₃ nanorods measuring in average 4.1 ± 1.2 nm in width and 20.2 ± 7.0 nm in length. These nanoparticles were successfully coated with polyvynilpirrolidone (PVP) to improve their biocompatibility. According to IR and DSC results, we achieved a successful ligand exchange to PVP, with a defined binding with the nanoparticle. We studied the nanoparticles’ stability in different biological media (water, culture medium, fetal bovine serum, and human plasma). The stability tests showed that the suspensions were more stable for media with higher protein components. This may aid in the ability of the nanoparticles to move through the bloodstream. We studied the nanoparticles’ cytotoxicity in human breast cancer cells (MCF7), over 72 hours of incubation. The coated nanoparticles did not evidence cytotoxicity up to 2500 g/mL. Also, we conducted radiosensitivity tests using a Co-60 source. We determined that the best condition for adequate radiosensitizing test is a dose of 2 Gy and a post-irradiation incubationtime of 48 hours. MCF7 cells that were incubated with a nanoparticle concentration of 50 µg/mL and irradiated showed a 38% increase in cell death when compared to cells that were only irradiated, without nanoparticles. Our results confirm that PVP-coated Bi₂S₃ nanoparticles are stable in many biological media, and that they are not-cytotoxic in a wide range of concentrations. When combined with gamma radiation, the nanoparticles increase the efficiency of radiotherapy. This outstanding results certainly grant hope for this line of research, to develop cancer treatments that have less seconday effects and a better quality of life for patients.

Use of nanotechnology in gene therapy to deliver therapeutic nucleic acid such as siRNA could open up a new avenue to cure genetic disorders. Lipid nanoparticles currently in use for several biomedical applications integrate cationic lipids in order to form complexes with nucleic acid cargo and enable their uptake by target cells. However, larger surface area and highly charged nature of these nanocarriers increase their interaction with host cells which might induce toxicity. While gene mediated off-target effects have been extensively studied, it is crucial to consider the carrier toxicity and side effects including immunological parameters. In the immune system, phagocytes can engulf foreign materials, therefore they are appropriate to screen for immunotoxicity by monitoring their functions in unspecific and specific immune responses.
In our study, we evaluated the function of primary bone marrow derived macrophages (BMDMs) in response to cationic nanostructured lipid carriers (cNLCs), which are positively charged (+45.8 mV). The effect of cNLCs was investigated in non-activated and IL-4 or LPS-activated BMDMs. We assessed the secretion of proinflammatory molecules including, IL-6, TNF-α, MCP-1, Nitric Oxide (NO). We also addressed the effect of cNLCs on metabolism by evaluating glycolytic activity (glycolysis and glycolytic capacity) and oxidative phosphorylation (basal respiration, proton leak, ATP production, maximal respiratory capacity, spare respiratory capacity). Our results showed that cNLCs remarkably enhanced the secretion of several molecules (IL-6, TNF-α, MCP-1, NO) as well as the energy flux in non-activated or LPS- or IL-4-activated BMDMs. Furthermore, we combined cNLCs with negatively charged siRNA at different N/P ratios (the ratio of positively-charged amine groups of cNLCs (N = nitrogen) to negatively-charged phosphate (P) groups of nucleic acid) (N/P = 8/4/2/1). Our experiments showed that reversing the surface charge prevent the effect induced by positively charged cNLCs on IL-6, TNF-α, MCP-1, and NO productions as well as on cellular metabolism (glycolysis and oxidative phosphorylation) of BMDMs. Our results highlight that reversing the surface charge of cationic lipid nanocarriers with an oppositely charged biomaterial, for instance nucleic acid, could attenuate immunostimulatory activities of the cationic nanocarrier. This issue needs to be addressed during the use of cationic nanocarriers in different biomedical applications, such as nucleic acid delivery for therapies or vaccines.
Finally, we propose to tune the nucleic acid load, hence the surface charge of lipid nanocarriers is critical for their therapeutic use, to prevent the alteration of immune cell response to positive charge stimuli. We also recommend to measure the zeta potential of the nanocomplexes, keeping in mind that charged nanocomplexes can alter the immune cell function which may lead to several side-effects.

This project has received funding from the European Union’s Horizon 2020 research and innovation program H2020 “NEWDEAL” (grant agreement No. 720905).

Binding antibodies onto a cell surface triggers antibody-mediated effector killing by innate immune cells through complement activation (CDC), antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent phagocytosis (ADCP), which is the mechanism of action of many monoclonal antibody (mAbs) therapeutics. As an alternative to mAbs, synthetic systems that can recruit endogenous antibodies, i.e. antibodies against small molecule haptens that are found in the blood of virtually every human being, from the blood stream to a cancer cell surface could be of great relevance. Herein, we explore antibody-recruiting polymers as a novel class of immunotherapy. These polymers are functionalized on their surface with multiple cell-binding and antibody-binding hapten motifs such as dinitrophenol and rhamnose. As cell surface binding motifs, we employ alkyl lipid and dibenzoylcyclooctyne motifs, for respectively hydrophibically-driven insertion into the phospholipid cell membrane or strain promoted alkyne-azide cycloaddition with azide motifs in the glycocalyx of cancer cells. In vitro on cancer cell cultures, we demonstrate that antibody-recruiting polymers allow for high avidity antibody binding and drive antibody recruitment to treated cells for several days and elicit robust innate immune effector killing.

Mitochondria are energy-producing organelles that exist within eukaryotic cells contained within a double phospholipid membrane. The inner membrane is composed of about 20% cardiolipin (CL), an unusual dimeric phospholipid in which a pair of phosphatidic acids are linked by glycerol to yield a molecule with four acyl chains. CL is the hallmark of energy transducing membranes and is implicated in a number of critical pathways in mitochondrial metabolism, regulation, and apoptosis. Abnormalities in CL composition profiles are linked to several diseases, including Barth syndrome, Tangier disease, heart failure, and neurodegenerative disorders [1]. The acridine orange derivative, 10-N-nonyl acridine orange (NAO), is a high-affinity probe that is 30 times more specific for CL than for other cationic phospholipids and is used as a stain for fluorescent visualization and quantification of mitochondrial CL [2]. Enhancing probe uptake by CL enables the use of lower dye concentrations and lowers cellular toxicity, but efforts to rationally engineer NAO to increase staining efficiency have compromised its phospholipid selectivity [3]. In this work, we have combined coarse-grained molecular simulation, alchemical free energy calculations, deep representational learning, and Bayesian optimization to perform high-throughput virtual screening of candidate organic molecules as CL dyes with superior uptake and selectivity than NAO. We consider as our search the >10¹¹ small organic molecules up to 400 Da molecular weight and use our computational platform to rationally traverse this space. Our analysis efficiently identifies novel high performing dye molecules and informs new understanding of the molecular properties responsible for good efficiency and selectivity. After just a few rounds of iterative molecular simulation and data-driven model building, we have identified three candidates predicted to have up to 4-fold superior uptake and 99 candidates with up to 34-fold superior selectivity relative to NAO. Future collaborative work is planned to synthesize and test the top compounds identified in the computational screen.

1. G. Paradies, V. Paradies, F.M. Ruggiero, and G. Petrosillo, Cells 8 (7) 728 (2019)
2. M.E. Rodriguez, K. Azizuddin, P. Zhang, S.-M. Chiu, M. Lam, M.E. Kenney, C. Burda, and N.L. Oleinick, Mitochondrion 8 (3) 237-246 (2008)
3. J. Zielonka, J. Joseph, A. Sikora, M. Hardy, O. Ouari, J. Vasquez-Vivar, G. Cheng, M. Lopez, and B. Kalyanaraman, Chemical Reviews 117 (15) 10043-10120 (2017)

Therapy based on personalized medicine has become a leading strategy to treat cancer. Small molecule drugs such as kinase inhibitors, which target key effectors of cancer signaling pathways, constitute a major component of this strategy. This class of drugs is diverse and each has different issues regarding pharmacokinetics, toxicity, and therapeutic index, especially in intracranial tumors due to the blood-brain barrier. Kinase inhibitors also inhabit a diverse chemical space, making it difficult to develop nanomedicines against the wide set of precision drug targets. We developed data science/machine learning-driven processes to predict and facilitate the encapsulation of diverse drug classes into nanoparticles based on the molecular structures of the drugs. These included quantitative structure-nanoparticle assembly prediction models for drug payload selection. We found molecular descriptors that are highly predictive indicators of nano-assembly and nanoparticle size. For the treatment of pediatric tumors with an intact blood-brain barrier, we developed a therapeutic nanoformulation capable of traversing the blood-brain barrier via transendothelial transport across tumor vasculature to improve kinase inhibitor therapeutic index.

Novel imaging and drug delivery methods are needed to understand the cellular and molecular events of cancer metastasis. We have developed a microfluidic imaging window (MFIW) platform that enables precise delivery of drugs, labeling agents, and cells during longitudinal intravital imaging of the tumor microenvironment. Intravital imaging with the MFIW platform enables in vivo evaluation of drug effects on tumors, and observation of cellular events, such as the intravasation of tumor cells into blood vessels. An important aspect of fluid delivery is to precisely control the fluid flow vector field and three-dimensional distribution of reagents in the target tissue. Using a dry film SU-8 photoresist we have implemented a novel photolithography process dubbed post-exposure lamination (PEL). Using PEL we have successfully integrated micro-nozzle structures with planar microfluidics, to significantly enhance three-dimensional control of fluid infusion. The PEL method enables control of the vertical and horizontal morphology of micro-nozzle structures, yielding nozzles that produce unique flow profiles. The micro-nozzle structure improves the convective transport of reagents into a projected delivery area that is 70% smaller than the projected delivery area of devices with a planar outlet. Confocal microscopy and computational fluid dynamic simulations are currently being used to assess the depth penetration and radial spreading of fluid in a tissue phantom, with initial results showing improved penetration depth and radial profile when micro-nozzle outlets are employed. Ongoing work is focused on localized delivery of transfection reagents to cells in three-dimensional culture, to demonstrate the therapeutic potential of the platform.

More than 2 million End Stage Renal Disease (ESRD) patients receive dialysis to sustain life, with this number likely to represent less than 10% of the actual need. In the United States alone, over 460,000 people are on dialysis. Conventional hemodialysis removes urea and other metabolic wastes from the body by running ~120 L of dialysate over hollow fiber dialysis membranes each session, which is typically 3-4 hours per session and thrice a week. The intermittent character of hemodialysis results in large fluctuations in blood metabolite concentrations. Observations show that long term survival in dialysis patients treated by extended hemodialysis are improved compared to conventional hemodialysis. Thus a portable dialysis machine that is working continuously would bring in significant health, quality of life and economic benefits.

To enable portable kidney dialysis for ESRD, the regeneration of the dialysate in a closed loop system is a primary critical technical barrier. Currently the ~120 L (kg) of dialysate per session, far exceeds usable weights for a portable system. We have developed an efficient photooxidation system based on hydrothermally grown TiO₂ nanowires, UV LEDs, and catalytic gas diffusion barriers to decompose urea from the dialysate at rates sufficient to remove daily production of urea at 15 g/day.

A photoelectrical decomposition cell with SiO₂/FTO/TiO₂ nanowire anode, 10 mM urea/0.15 M NaCl electrolyte, and 4 mg/cm² Pt black loaded carbon paper cathode was characterized for urea decomposition efficiency. Under 4 mW/cm² illumination of the 365 nm LED with 40% quantum efficiency, the device yielded a photocurrent density of ~ 1 mA/cm² in a dialysate simulant of corresponding to 40% quantum efficiency in urea decomposition per incident photon. From performance parameters, a feasible portable device with ~0.23 m² active area and a current draw of 11 A is able to decompose a daily 15 g urea production sufficient to regenerate dialysate. Also high selectivity (80%) for urea decomposition over Cl2 formation is shown. For comparison, prior reports in literature [J. Photochem. Photobiol. AChem, 2009, 205, 168] for urea fuel cells of agricultural waste, > 5000 A would be required. Improvements reported here are based on nanowire microstructure to efficiently separate electron holed pairs and efficiently adsorb incident UV irradiation.

Introduction: Filamentous nanoparticle (FN) refers to a polymeric self-assembly with a high aspect ratio, nanometer scale in diameter and micrometer scale in length. FN has great potential in drug delivery because it shows a longer circulation time, low phagocytic cell uptake, and longer retention in tissues as compared to spherical counterparts. FN is produced with amphiphilic block-co-polymers, which interact with each other via directional and reversible noncovalent interactions. For biomedical applications, FN is usually prepared with a peptide-drug conjugate, in which a drug is part of the building block of the FN. We report a lithocholic acid-polyethyleneimine conjugate (lp) that can form FN in the presence of hydrophobic drugs, with no need of the drug being part of the polymer, and serve as their carrier for local drug delivery. We characterized the properties of FN, studied the conditions that enable FN formation, and tested the feasibility of using FN for local drug delivery.
Methods: FN was prepared by the membrane hydration method. Various small molecule drugs or model compounds were screened to test the ability to form FN with lp. Retrospective quantitative structure-property relationship (QSPR) analysis was performed by Alvadesc software to identify common structure features of FN formers. The formation of FN and FN hydrogel was studied varying the conditions such as aging time, temperature, lp/drug ratio, and drug concentration. Tissue retention of paclitaxel FN labeled with DiR was monitored by intratumoral injection of FN to CT26 colon tumors in Balb/c mice and whole-body fluorescence imaging.
Results: Out of 19 drugs screened, 6 drugs formed FN with lp, 4 formed short filaments, and 9 did not form FN. QSPR analysis revealed that FN formers had amido bond in common. The diameter of FN was measured to be 26.3 ± 4.3 nm, and the average length ranged from 1 to 2 μm depending on the type of drug and the weight ratio of lp to drug. The rate of FN formation increased with temperature. Paclitaxel-loaded FN formed a hydrogel at 37.8 mg/mL or higher, likely due to the physical entanglement of FN. FN injected at 4.46 mg/mL to subcutaneous CT26 tumors persisted for more than 8 days showing 80% of the initial FN signal in the tumor on day 8. In contrast, spherical counterparts diffused away with only 50% FN remaining after day 1 and all disappearing after 1 week. This result indicates the potential of FN to serve as a local drug delivery system.
Conclusion: Lithocholic acid-polyethyleneimine conjugate formed FN with a group of hydrophobic drugs. The drug-loaded FN can serve as a local depot of the drug in tumors. The lithocholic acid-polyethyleneimine conjugate can be combined with various hydrophobic drugs with amido bond structure to form FN and FN-based hydrogel for systemic and local drug delivery.

DNA and RNA can manipulate the expression of any gene, making these molecules promising drugs. However, whether the drug is comprised of DNA, siRNA, mRNA, lncRNA, or another nucleic acid, it is limited by one problem: drug delivery. Chemists design thousands of distinct nanoparticles to deliver DNA or RNA to the desired cell type. However, after nanoparticles are synthesized, their ability to deliver drugs is evaluated using in vitro systems devoid of a liver, kidney, spleen, immune system, pulsatile blood flow, and other selection pressures known to affect nanoparticle delivery in vivo.
We have designed a series of increasingly advanced DNA barcoding platforms to quantify how thousands of nanoparticles deliver nucleic acids in vivo. Our goal is to quantify how up to 300 nanoparticles deliver DNA, mRNA, ASOs, or siRNA into up to 30 cell types, all in a single animal. To analyze these large in vivo drug delivery datasets, we have also developed an open source bioinformatics pipeline to iteratively ‘evolve’ nanoparticles that target cells in vivo. Using this high throughput, iterative, in vivo approach, we have identified nanoparticles with tropism to many novel cell types, in many different tissues.

Uncontrolled systemic inflammatory immune triggering hampers clinical translation of many classes of small molecule immunomodulators for vaccine design and cancer immunotherapy. Potent small molecule agonists of pathogen recognition receptors such as toll liker receptors and agonists of STING have been discovered, but like many other small molecule drugs, they are prone to rapid distribution throughout the body, thereby losing sitespecific activity and causing unwanted systemic inflammation.
Noncovalent albumin binding has proven a powerful strategy for lymphoid delivery. By taking advantage of the inherent serum protein binding property of lipid motifs and their tendency to accumulate in lymphoid tissue, we designed lipid amphiphile conjugates that show robust lymphatic translocation by hitch hiking onto albumin molecules in the interstitial flow from the injection site to draining lymphoid tissue. By conjugating a small molecule immunomodulator (exemplified in our work by an imidazoquinoline TLR7/8 agonist) to lipid amphiphiles through a degradable linker we obtain well water soluble prodrugs that, upon subcutaneous or intramuscular administration provoke potent lymph node immune activation but suppress systemic overstimulation of the immune system.
Lipid conjugation of potent small molecule immunomodulators has shown to reduce systemic toxicity but comes at a cost of a dramatic reduction in biological activity due to the formation of an insoluble depot. Our approach results in a well water soluble lipid amphiphile prodrug, showing high biological activity in vitro and in vivo in mouse models.

With profound advances in immune-oncology, cancer immunotherapy is now considered the fourth pillar of cancer therapy, joining the ranks of surgery, radiotherapy, and chemotherapy. For some cancers, including advanced non-small cell lung cancer, combination immunotherapy is FDA-approved as the frontline therapy, showing promise for applying immunotherapy to a wide range of advanced cancers. Currently, immune checkpoint blockade involves systemic administration of monoclonal antibodies, which can cause off-target side effects by inducing activation of self-antigen reactive T-cells. The combination of multiple immune checkpoint blockers generally improves the clinical responses; however, this can lead to severe immune-related adverse events that result in clinical manifestations of dermatitis, colitis and hepatitis. Thus, to fully realize the potential of cancer immunotherapy, approaches are needed to amplify anti-tumour T-cell immune responses, to convert cold tumours into hot tumours and to sensitize tumours to immunotherapies with minimal off-target toxicity and immune-related adverse events. Here, we highlight new opportunities for combination immunotherapy based on nanomedicines that are well poised tackle the challenges faced by the field of cancer immunotherapy. We present biomaterial-based strategies for amplifying anti-tumor immune responses and sensitizing tumors to immunotherapies in a safe and effective manner. Briefly, we show that lipid-based nanodiscs can efficiently co-deliver antigen and immunostimulatory molecules to draining lymph nodes and elicit potent CD8+ cytotoxic T lymphocyte responses directed against tumor antigens, leading to substantially enhanced anti-tumor efficacy in multiple murine tumor models, including colon carcinoma, melanoma, and glioblastoma multiforme. In a second research thrust, we have shown that this nano-platform can deliver chemotherapeutic agents in a synergistic manner with immune checkpoint blockers. In a third research thrust, we are developing new biomaterials for in situ modulation of the gut microbiome for regulation of local and systemic immune responses. We will share our latest results showing the therapeutic potential of our gut modulation approach in the context of combination cancer immunotherapy. Owning to the facile manufacturing process, robust therapeutic efficacy, and good safety profiles, our biomaterial-based approaches may offer powerful and convenient platforms for combination cancer immunotherapy.

In the last decades, great attention has been paid to developing of nanoscale vehicles for drug delivery. Incorporation of drug molecules into nanocarriers allows to overcome poor water solubility of hydrophobic drugs as well as increase stability against hydrolytic degradation of hydrophilic ones. Moreover, nanoparticulate drug formulations can act in a targeted and prolonged manner enhancing the efficacy of treatment, e.g. cancer treatment. Platinum-based complexes (cisplatin, carboplatin, oxaliplatin etc.) are widely used chemotherapeutics agents for the treatment of various types of cancer. Oxaliplatin ((trans-R,R-cyclohexane-1,2-diamine)oxalatoplatinum(II)) is the third-generation platinum complex that was designed to overcome cellular resistance to cisplatin and carboplatin. Oxaliplatin shows higher solubility and less toxicity than cisplatin. It is used as a standard treatment for colorectal cancer. Moreover, oxaliplatin can be active against refractory ovarian cancer, germ-cell cancers, non-small cell lung cancer etc. Nevertheless, its low water solubility, short half-life in the bloodstream and non-selective biodistribution reduces the effective dose of oxaliplatin in the targeted tissues and enhances the systemic toxicity. The design of new types of nanocarriers for delivery of oxaliplatin is of high interest. We believe that P(D,L)LA-b-PEG nanoparticles is a promising platform due to its flexibility and successful track record as a nanocarrier for development of targeted anticancer drug formulations.
Biodegradable drug-free and oxaliplatin-loaded mPEG₁₁₃-b-P(D,L)LA_n nanoparticles were prepared by a simple nanoprecipitation technique. The effect of hydrophobic block length on the structure, size, morphology and drug loading content of mPEG₁₁₃-b-P(D,L)LA_n nanoparticles was investigated. It was observed that in aqueous solution mPEG₁₁₃-b-P(D,L)LA_n copolymers, where n = 62 – 173 monomer units, form spherical nanoparticles with hydrodynamic diameters ranging from 32 to 56 nm. The “core-corona” structure of the block copolymer nanoparticles was confirmed by SAXS. Tailoring of P(D,L)LA block length results in variation both core-corona interface area and tethering density of hydrophilic PEG chains on the surface of P(D,L)LA core of the mPEG₁₁₃-b-P(D,L)LA_n nanoparticles, which affects oxaliplatin loading content. An increase in P(D,L)LA block length from 62 to 173 monomer units results in a decrease in core-corona interface area from 2.7x10²⁰ to 1.5x10²⁰ nm²/g and tethering density of PEG chains from 1.6 to 1.0 nm^-2 and a reduction in the oxaliplatin loading content from 3.8 to 1.5% wt./wt. Thus, we suppose that oxaliplatin is adsorbed on the core-corona interface of the mPEG₁₁₃-b-P(D,L)LA_n nanoparticles. SAXS measurements revealed that oxaliplatin loading does not affect the size and structure of the block copolymer nanoparticles.
The size and structure of polymeric nanoparticles are crucial characteristics that should be considered in the design of targeted nanoformulations of anticancer agents. The developed formulations of oxaliplatin can be considered as promising candidates for treatment of various types of cancer. In vitro tests were performed in order to compare its efficacy and toxicological profile with pure oxaliplatin.
This research was funded by the Russian Science Foundation, grant number 18-73-10079.

Colorectal Cancer (CRC) is the fourth leading cause of cancer-related deaths with few available treatment options. Common treatment techniques are surgery and chemotherapy. Among the available chemotherapeutic drugs, oxaliplatin is a cornerstone for the treatment of CRC. As an adjuvant technique, hyperthermia i.e. an elevated temperature (39–42 °C), has been shown to increase chemotherapy treatment outcomes clinically. Hyperthermia increases the drug uptake by affecting cell membranes and produces drug-induced DNA damage leading to enhanced tumor cell death. It has been demonstrated that the synergistic effect of hyperthermia with oxaliplatin can treat CRC. One of the major disadvantages is, due to the intermittent exposures of oxaliplatin the cells become chemo-resistance complicating the treatment procedure. To improve the precision of the technique, instead of using a bulk carrier fluid along with a heat exchanger in the traditional hyperthermia delivery, photothermal nanoparticles can be used to deliver more specific and effective hyperthermia.
In our laboratory, semiconducting polymer nanoparticles have been used as photothermal agents to ablate cancer cells. Recently, variable molecular weight nanoparticles (VMWNPs) produced from the oligomer and high MW segments of a single polymer poly[4,4-bis(2-ethylhexyl)–cyclopenta–[2,1-b:3,4-b″]–dithiophene-2,6-diyl-alt-2,1,3-benzoselenadiazole-4,7-diyl] (PCPDTBSe) were demonstrated as a promising photothermal agent for the ablation of breast cancer. VMWNPs generated heat upon 800 nm laser irradiation and produced fluorescence emission at 825 nm upon excitation at 550 nm. Therefore, VMWNPs can be used to detect CRC through near infra-red fluorescence imaging and hyperthermia delivery. Here we have explored the synergistic effect of oxaliplatin and hyperthermia generated through VMWNPs to augment chemotherapy treatment of chemo-sensitive and chemo-resistant CRC cells.
The process of hyperthermia generation using VMWNPs was optimized, and it was found that a 25 µg/ml concentration of VMWNPs could produce hyperthermia (39–42 °C) effect upon 800 nm (3W) laser exposure for 20 seconds. Various concentrations of VMWNPs were incubated with mouse-derived CRC CT-26 cells for 24 hours and no cytotoxic effect was observed up to 100 µg/ml. Whereas upon incubation of oxaliplatin with increasing concentrations there was a 50% decrease in cell viability at 300 µM concentration was observed. To monitor the thermal dose, first, the chemo-sensitive and chemo-resistant CRC cells were incubated with varying concentrations of oxaliplatin and kept at 42 °C for two hours. An 80% and 75% decrease in cell viability was observed for chemo-sensitive cells and chemo-resistant cells respectively. To observe the effect of VMWNPs induced hyperthermia, CRC chemo-sensitive cells were incubated with oxaliplatin and VMWNPs followed by laser exposure (800 nm, 3W, 20 secs) in three cycles. A 63% reduction in cell viability was observed for VMWNPs generated hyperthermia. This result confirmed that VMWNPs could successfully deliver hyperthermia to augment chemotherapy. In the future, oxaliplatin, and hyperthermia using VMWNPs will be utilized to treat chemo-resistant CRC cells.

Protein-protein interactions (PPIs) dictate most biological processes, including those responsible for carcinogenesis. Targeted therapeutics acting on these oncogenic PPIs have naturally become the focus of novel drug discovery. Currently,one small molecule PPI inhibitor, Venetoclax (AbbVie) is FDA approved to treat a number of hematologic malignancies through its inhibition of an antiapoptotic protein, BCL-2. However, considering that there are ~650,000 human PPI s as potential targets, the development of other PPI inhibiting drugs has been lagging due to the off-target and delivery issues. For example, a PPI inhibitor, ABT-263, although possessing potent nanomolar binding affinity to BCL-2, can lead to sever thrombocytopenia when administered systemically, which is due to the off-target BCL-XL inhibition in platelets. This is limiting its therapeutic index. We have developed a nanocarrier system, targeted polymersome (PSOM) nanoparticle, made of biocompatible poly(ethylene glycol)-disulfide-poly(propylene sulfide) (PEG-SS-PPS) with surface conjugated anti-CD19 F(ab) antibody fragments (aCD19 Fabs) as targeting motif to B cell lymphoma. PSOMs are fabricated by flash nanoprecipitation (FNP) and extrusion resulting in uniform vesicular morphology and size distribution (~130 nm in diameter). Moreover, ABT-263 was successfully encapsulated by PSOM at an efficiency over 90%, which increases the solubility of the lipophilic ABT-263 to over 10mM in PBS. From in vivo platelet-counting experiment, the intravenous administration of PSOM encapsulated ABT-263, in contrast to orally administered non-encapsulated drug, led to the mitigated thrombocytopenia in mice. Importantly, flow cytometric analyses on a panel of human CD19 positive diffuse large B cell lymphoma cell lines demonstrated specific uptake of aCD19-PSOM nanoparticles. We believe the results indicate the great promise of in vivo targeted delivery of ABT-263 by PSOM to B cell lymphoma.

The field of acoustic droplet vaporization (ADV) was introduced nearly 20 years ago to embolize tissues non-invasively. ADV used 1-5 µm perfluorocarbon (PFC) droplets and converted them into large gas bodies in the feeding artery with ultrasound to embolize tissues downstream. Despite its potential clinical impact, the ADV field has not advanced. The manufacture of nanodroplets (ND) using PFC that boils near 0 ^oC to ease vaporization, was introduced 10 years later to rescue ADV, and to capitalize on the nanosize ND for tumor detection. Despite this advance, the field remains stagnant because of critical efficacy and safety challenges hindering its translation and widespread use. Some important challenges include the energetics required to cavitate the PFC into gas, and the resultant rapid expansion that could injure tissues.
We recently discovered that PFC nanobubbles (NBs) and microbubbles (MBs) inflate when exposed to superheated PFC NDs. While in vitro and in vivo preliminary data confirmed the proof of concept, the translational success of our novel approach will rely on our ability to control bubble inflation to safely improve tumor detection when NBs are used or occlude tumor microvascular when MBs are used. We showed that both rate and degree of expansion is affected by the type of PFC used, ND and NB/MB size and type of lipid emulsifier that affect both interfacial tension and the ability of bubbles to expand. We showed that MBs with a stiff polymeric shell made of denatured albumin when exposed to liquid PFC NDs expand more rapidly, but to only half the diameter of typical phospholipid-based MBs, encouraging us to optimize shell formulation to limit inflation.
As this non-invasive platform can be used to perform chemoembolization as is currently done to treat liver tumors but without the need for angiography or prior tumor visualization, one of our current goal is to load chemotherapeutics in NDs or MBs and promote drug release in only tissues expressing the receptor of interest, analogous to trans-arterial chemoembolization. However, since this approach is not image guided, tumors do not need to be visible pre-treatment. We believe that these accomplishments show promise and potential to translating our novel approach to the clinic while creating new paradigms for early detection and treatment of cancer. This talk will be focused on the impact of formulation on MB growth and translational opportunities.

Interventions that engage the power and specificity of the immune system have enormous potential to improve human health, yet the efficacy, safety, and utility of many promising immunomodulatory agents is limited by critical drug delivery barriers. This talk will describe current research in my laboratory focused on engineering nanomaterials for intracellular delivery of immunomodulatory proteins, nucleic acids, and small molecules. Specifically, I will discuss our recent efforts in developing environmentally-responsive “smart” materials that open access cytosolic immune surveillance pathways, including STING and RIG-I, and how we are leveraging these platforms to enhance responses in cancer immunotherapy and augment the efficacy of subunit vaccines against respiratory viral pathogens.

The development of an efficient phototherapeutic system is highly desirable but still a big challenge, because of its significant limitations at tissue penetration. NIR photo upconversion can be an effective key to assist electromagnetic energy penetrating deep tissue into most tumor locations. Herein, a novel 808 nm triggered upconverting system was designed and applied for NIR triggered drug release and simultaneous photodynamic therapy. A core-shell NaYF₄:Nd/Yb@NaYF₄:Yb/Er nanocrystal was rationally synthesized, exhibiting photoluminescence emissions at the wavelength around 538 nm, under NIR laser radiation. The UCNPs were surface modified with silanization and amine terminals decoration. Subsequently, via a carbodiimide coupling reaction, UCNPs were grafted with bifunctionalized C₆₀ derivative, as the photosensitizer. Then the C₆₀ coated UCNPs were further conjugated with chemotherapy model drug Doxorubicin (Dox), through a covalent oxalyl linkage. Notably, under NIR laser irradiation, singlet oxygen was effectively generated from an upconverting photodynamic combination of UCNPs and C₆₀. And Dox got a controlled release from cleavage of the peroxide sensitive oxalyl bridge. The drug release and therapeutic efficacy were investigated in vitro on breast cancer cell lines MCF-7 and MDA-MB-231 by various microscopic and biochemical studies under a significantly mild NIR irradiation and low dosage of the treatment. The combination of controlled release for localized chemotherapy with simultaneous photodynamic treatment has great potential for clinical photo cancer therapy.

Photothermal therapy is promising for cancer treatment as it is noninvasive and localized. The way to treat tumors is elevating the temperature through conversion of radiation energy to heat under near-infrared (NIR) light. To further enhance the therapeutic effect of cancer treatment, anti-cancer drugs are released to the photothermally-heated tumors. The chemo-photothermal therapy can synergistically enhance the efficacy of tumor treatment. For the chemo-photothermal therapy, it is required to design drug carriers that provide photothermal heating and on-demand release of drugs. At the same time, the carriers should have high payload and high efficiency of encapsulation for the drugs. However, there have been few reports on such an advanced carrier for the synergistic chemo-photothermal therapy.
Herein, we report a pragmatic microfluidic approach to produce monodisperse microcapsules for chemo-photothermal therapy. The microcapsules contain highly concentrated polydomapine (PDA) nanoparticles and drugs in their aqueous lumens, enclosed by thermo-responsive membrane. The microcapsules are templated by water-in-oil-in-water (W/O/W) double-emulsion droplets. With a glass capillary microfluidic device, monodisperse double-emulsion droplets are produced to have the innermost water drops containing PDA nanoparticles and drug and the outer oil drops of photocurable monomer, phase-change materials (PCM), and molecular compatibilizer. As a continuous phase, an aqueous solution of high concentration of salt and surfactant is used to make a hypertonic condition. The double-emulsion drops shrink downstream as water in the innermost drops is pumped out through the oil shell by the osmotic-pressure difference, which concentrates the PDA nanoparticles and drugs during the flow. The resulting droplets are irradiated by ultraviolet (UV), which photopolymerizes the monomer in the oil shell. In the presence of a molecular compatibilizer between the monomer and PCM, the polymerization does not cause any macrophase separation, resulting in a composite membrane with continuous microscopic PCM domains. The polymer framework in the membrane provides mechanical stability, whereas PCM domains provide channels for transmembrane transport at the temperature above a melting point for molecules dissolvable in the molten PCM. To use the microcapsules for in-vivo drug release, tetradecanol with melting point of 38°C is chosen as a PCM so that the microcapsules retain drugs at body temperature while secreting them when temperature is elevated; many anti-cancer drugs are dissolvable in organic solvents as they are not highly polar.
The microcapsules show a rapid temperature increase under the irradiation of 808 nm laser as highly-concentrated PDA nanoparticles in the lumen provide a high photothermal conversion efficiency. Therefore, the PCM in the membrane is molten under the laser irradiation and the drugs in the lumen is gradually released out. As the heating is highly localized, the microcapsules are cooled down as the laser is off, holding up the release. Therefore, the release as well as local temperature can be controlled in a programmed manner by controlling the duration and interval of stepwise irradiation. With the microcapsules, we successfully treat breast cancers using animal models through chemo-photothermal therapy where palbociclib is used as an anti-cancer drug. The combination of drug and local heating shows higher efficacy of the treatment than the photothermal therapy or chemotherapy used only. We believe the resultant microcapsules with synergistic chemo-photothermal effect are highly attractive in use of advanced chemo-photothermal therapy.

Intratumoral injection of adenovirus (Ad) into diseased tissues remains a conventional route for viral gene delivery. Nonetheless, locally administered virus often disseminates to the surrounding nontarget tissues from the injection site, leading to poor localization and retainment of the virus in tumor tissues. Rapid induction of antiviral immune response against Ad also contributes to poor longevity of virus at tumor site. To address these limitations, diverse array of nanomaterial-based delivery systems for oncolytic Ad have been investigated to improve intratumoral retainment and accumulation of virus at tumor tissues, while attenuating nonspecific sequestration into healthy organs. Importantly, nanomaterial coating on the surface of viral capsid can facilely endow novel properties to the resulting nanohybrid complex, which cannot be achieved through genetic engineering of the virus genome. For instance, masking of viral capsid with nanomaterials can endow “stealth” ability to the virus, enabling it to evade the detection by host immune system and subsequently attenuate antiviral immune response against Ad, ultimately prolonging virus’ biological activity at tumor tissues. Importantly, optimization of nanohybrid system can ensure that oncolytic Ad’s ability to induce robust antitumor immune response can be preserved while selectively attenuating induction antiviral immune response, thus resulting in improved potency and safety profile. Another advantage of nanohybrid system is that diverse array of cancer targeting moieties, like antibodies and peptides, can be easily incorporated on the surface to redirect the virus tropism to wide range of tumors expressing complementary receptors. Sustained or controlled release of the virus at tumor sites can also be achieved by nanomaterial-based delivery system to improve and prolong virus retention at tumor sites. Collectively, nanomaterial-based delivery systems for oncolytic adenovirus is a promising strategy to overcome many of the clinical hurdles to achieving optimal virus delivery to tumor tissues.

Biomaterials can improve the safety and presentation of therapeutic agents for effective immunotherapy, and a high level of control over surface functionalization is essential for immune cell modulation. Here, I will discuss biocompatible immune cell engaging particles that use synthetic short DNA as scaffolds for efficient and tunable protein loading. The use of DNA scaffolds allows for ratiometric control and high-density biomolecular loading. Moreover, intratumorally injected immune cell engaging particles presenting a high density of priming antigens activated CAR-T cells drove local tumor clearance while sparing uninjected tumors in immunodeficient mice. The ratiometric control of costimulatory ligands and the surface presentation of a cytokine on immune cell engaging particles were shown to significantly impact human primary T cell activation phenotypes. I will also discuss ways to use nanostructured biomaterials to selectively capture endogenous cytokines in order to preferentially engage specific immune cells. These modular and versatile biomaterial platforms can provide new opportunities for immunotherapies.

It has witnessed that the rapid development on precision design and fabrication of intelligent next generation nanomedicines hold great potential to revolutionize the current landscape of drug development. It is also clear that tumor microenvironment plays critical roles on either promotion or restriction on primary tumor rapid growth and metastasis. Those achievements have made targeting and regulation of tumor microenvironment via responsive nanomedicines a feasible and fruitful strategy, to improve the therapeutic outcomes for cancer treatment. This presentation will feature our recent development on using biomolecule-based nanostructures as intelligent nanomedicines to regulate tumor microenvironment to block tumor microvessels or re-store the homeostasis of tumor stroma. Robotic molecular systems have great potential as intelligent vehicles to enable the delivery of various potent molecules, which otherwise never could be used as therapeutics due to numerous limitations. Yet, achieving in vivo, precise molecular-level, and on-demand targeting and delivery has proven extremely challenging. We developed nanorobotic systems for targeted cancer therapy, programmed to transport molecular payloads and cause on-site tumor infarction. Given the robust self-assembly behavior, exceptional designability, potent antitumor activity and minimal in vivo adversity, the development represents a promising strategy for precise drug design for cancer therapeutics.

References
Yinlong Zhang, Xuexiang Han, Guangjun Nie*, Responsive and activable nanomedicines for remodeling the tumor microenvironment, Nature Protocols, in press
Shaoli Liu, Qiao Jiang, Zhao X, Zhao R, Wang Y, Wang Y, Liu J, Shang Y, Zhao S, Wu T, Zhang Y, Guangjun Nie, Baoquan Ding*, A DNA nanodevice-based vaccine for cancer immunotherapy, Nature Materials. 2020 Sep 7. doi: 10.1038/s41563-020-0793-6.
Suping Li, Yinlong Zhang, et al, Guangjun Nie, Combination of tumour-infarction therapy and chemotherapy via the co-delivery of doxorubicin and thrombin encapsulated in tumour-targeted nanoparticles. Nature Biomedical Engineering, 2020 Jun 22. doi: 10.1038/s41551-020-0573-2.
Xin Zeng, Jie Sun, Suping Li, Jiyun Shi, Han Gao, Wei Sun Leong, Yiqi Wu, Minghui Li, Chengxin Liu, Ping Li, Jing Kong, Yi-Zhou Wu, Guangjun Nie, Yuming Fu & Gen Zhang, Blood-triggered generation of platinum nanoparticle functions as an anti-cancer agent, Nature Communications, 2020, 11, 567.
Xuexiang Han, Yiye Li*, Xiao Zhao, Yinlong Zhang, Xiao Yang, Yongwei Wang, Ying Xu, Ruifang Zhao, Gregory J. Anderson, Yuliang Zhao*, Guangjun Nie*, Reversal of pancreatic desmoplasia by re-educating stellate cells with a tumour microenvironment-activated nanosystem, Nature Communications, 2018, 9, 3390.
Suping Li, Qiao Jiang, Shaoli Liu, Yinlong Zhang, Yanhua Tian, Chen Song, Jing Wang, Yiguo Zou, Gregory J Anderson, Jing-Yan Han, Yung Chang, Yan Liu, Chen Zhang, Liang Chen, Guangbiao Zhou, Guangjun Nie*, Hao Yan*, Baoquan Ding*, Yuliang Zhao*, A DNA nanorobot functions as a cancer therapeutic in response to a molecular trigger in vivo, Nature Biotechnology, 2018, 36, 258-264.
Suping Li, Yinlong Zhang, Jing Wang, Ying Zhao, Tianjiao Ji, Xiao Zhao, Yanping Ding, Xiaozheng Zhao, Ruifang Zhao, Feng Li, Xiao Yang, Shaoli Liu, Zhaofei Liu, Jianhao Lai, Andrew K. Whittaker, Gregory J Anderson, Jingyan Wei, Guangjun Nie*, Nanoparticle-mediated local depletion of tumour associated platelets disrupts vascular barriers and augments drug accumulation in tumours, Nature Biomedical Engineering, 2017,1, 667–679.

Of the clinically approved nanotechnologies in 2019, none are indicated for non-cancerous neurological disease, which represents 13% of the global disease burden. Therefore, our lab has worked to develop tools that inform how we can more effectively treat the diseased brain, using nanotechnology as both a probe and as a therapeutic delivery vehicle. When delivery limitations of a nanoparticle platform are better understood, an optimal formulation can be engineered and evaluated for therapeutic efficacy in clinically relevant animal models of brain disease. We focus on developing nanoparticle-based therapeutic approaches for improved neurological outcomes in perinatal brain injury. We use therapeutic nanoparticle platforms that are polymer-based, incorporate materials that are FDA approved, and have been utilized extensively in adult populations but have not been used in children or newborns. We identify drugs that can affect multiple pathways and have a solubility and/or delivery problem - that is, the drug is either not soluble in aqueous solutions or it is not able to reach its target site in high enough concentrations to be effective. We use a range of in vitro, ex vivo, and in vivo models to screen and evaluate efficacy of nanotherapeutic platforms. Our ex vivo cultures include stimuli that replicate aspects of in vivo injury conditions, such as oxygen-glucose deprivation, glutamate toxicity, or exposure to lipopolysaccharide. In vitro and ex vivo, we test for toxicity and dose response; ex vivo, we evaluate regionally dependent cellular uptake and downstream mechanistic effects of the nanotherapeutic platform; in vivo, we quantify biodistribution and measure improvements in gross injury and neuropathology in response to treatment. We have shown curcumin-loaded polymer nanoparticles could reduce brain injury via gross injury analysis and neuropathology in neonatal rats with hypoxic-ischemic (HI) brain injury. We expanded on this work to show that superoxide dismutase can be an effective treatment against HI injury in cultured rat brain slices, and screened promising preclinical therapeutics in a ferret brain slice model of HI injury. Importantly, we’ve furthered our targeting capabilities by showing how the (1) disease severity alters nanoparticle-cellular interactions, (2) formulation conditions can impart pathology-specific toxicity, and (3) surfactant used to stabilize polymer formulations can direct site-specific cell-specific uptake in the neonatal brain.
In this talk, we demonstrate the importance of using multiple platforms to evaluate nanotherapeutic behavior in the brain, and the impact formulation methods can have on polymeric nanoparticle effect and fate in the brain. Further, we show that we can engineer more effective nanotherapeutics for the treatment of neurological disease with a specific emphasis on the neonatal and pediatric populations, which are vastly underserved by technology development.

Hydrogen sulfide (H₂S), a gaseous signal-transmitter molecule (gasotransmitter), has pivotal roles in the human body by regulating physiological and pathological processes such as angiogenesis, inflammation and neurotransmission. With the discovery of the biological significance of H₂S, the potential for H₂S-based therapies has attracted growing attention. However, the lack of H₂S delivery systems that enable controlled release of H₂S at the site of interest limits its biomedical applications. One common approach is to use H₂S donor compounds, such as anethole dithiolethione (ADT) derivatives, which generate H₂S under physiological conditions. However, the uncontrolled rate of H₂S release and toxic side effects of the donor compounds and/or their decomposition byproducts remain as the major problems associated with the use of small H₂S donor compounds. To address this issue, we developed polymeric micelles carrying H2S-donating ADT groups (H₂S donor micelles), which release H₂S in a sustained manner under physiological conditions. Here, we report design, synthesis and characterization of polymeric H₂S donor micelles. The proangiogenic activities of these micelles were also evaluated in the in vitro cell culture assays as well as the in ovo chick chorioallantoic membrane (CAM) assay.

Molecular imaging is recognized as a key technology for theragnosis which is known as an emerging paradigm for future personalized medicine. Peptide which have been used as a useful carrier for drug delivery system could also be served as an excellent tool for molecular imaging. Approaches to combine these two important functionalities all together in a peptide conjugate are proposed in this presentation. Recent advances in biotechnology have contributed to the development of various peptide conjugates that enable targeted delivery of imaging agents as well as therapeutic agents for biomedical applications. Compared to small molecules (e.g., imaging agents and therapeutic agents), peptide conjugates possess unique characteristics such as high target-specificity and multi-functionality. As a result, peptide conjugates hold great potential in the future biomedical field such as molecular imaging, diagnostics, and the drug delivery system. Moreover, combination of both imaging agents and therapeutic agents within a single peptide conjugates, often referred to as theragnostics, make it possible to provide useful information in drug developments. Theragnostic peptide conjugates allow in vivo real-time imaging of the diseased site, monitoring the biodistribution of drug and determining the optimal therapeutic efficacy following treatments. These features can allow clinicians to select optimal therapeutic options for personalized medicine. In recent studies, we developed epidermal growth factor receptor (EGFR) and CD 47 receptor-specific self-quenched imaging probes, which can emit fluorescence (activate) via de-quenching reaction in lysosome, resulting in showing target-specific fluorescence signal in vitro as well as in vivo condition. This presentation will highlight our recent advances that have been made in the development of multifunctional peptide conjugates and the applications into theragnosis.

Nucleic acids have paved new avenues for the development of therapeutic interventions against a spectrum of diseases; however, their clinical translation is limited by successful delivery to the target cells. To solve this problem, we fabricated cationic lipid-assisted nanoparticle (CLAN) by double emulsion method. In our initial design, the CLAN system was constructed using PEG-b-PLA and BHEM-Chol, which achieved >95% encapsulation efficiency of siRNA. Using CLAN, we and collaborators delivered siRNA, CpG and other nucleic acids for cancer and HBV treatment. To further improve its therapeutic efficacy, we synthesized a tumor pH-labile linkage-bridged block copolymer of poly(ethylene glycol) with poly(lacide-co-glycolide) (PEG-Dlink_m-PLGA) to prepare DCLAN_PLGA for siRNA delivery. The PEG surface layer of DCLAN_PLGA can be detached in response to the tumor acidic microenvironment to facilitate cellular uptake, and the siRNA was rapidly released within tumor cells due to the hydrophobic PLGA layer. Thus, PEG-Dlink_m-PLGA-based CLANs exhibited an enhanced siRNA delivery efficacy. Most recently, to rationally design the delivery system of CRISPR-Cas9 genome editing tools, we constructed a library of CLANs with different properties (e.g. surface PEG density, zeta potentials, cationic lipid types) by adjusting the weight and types of the cationic lipid and incorporating the homopolymer PLGA. After systemic administration, the uptake of these CLANs by different immune cells was evaluated, and the optimal CLANs were screened. Using this strategy, we efficiently delivered CRISPR-Cas9 into macrophages, neutrophils, B cells and dendritic cells for treating inflammatory diseases, type II diabetes, autoimmune diseases et al.. In summary, our CLAN is an effective platform for developing the delivery systems of siRNA, CRISPR-Cas9 and other nucleic acids.

Purpose. Polymeric vectors have been extensively studied to function as gene delivery systems, but their widespread applications have been restricted by inefficient in vivo delivery of therapeutic nucleic acids. In our studies, we investigate local and sustained delivery of nucleic acids making use of a polyplex-containing injectable hydrogel.

Methods. Complexes are formed between nucleic acids and cationic poly(2-dimethylaminoethyl methacrylate) (PDMAEMA)-based polymers (referred to a polyplexes), followed by the encapsulation of the polyplexes in a thermosensitive hydrogel. The designed triblock copolymer (PNIPAM-PEG-PDMAEMA, NPD) used for polyplex formation combines multiple functionalities, including cationic properties, needed for complexation with nucleic acids, and thermosensitive properties to anchor the polyplexes in the thermosensitive hydrogel. The physico-chemical properties of siRNA complexed to the multiresponsive polymer were evaluated and compared with polyplexes made with a non-thermosensitive polymer (PEG-PDMAEMA, PD) in 2D and 3D multicellular tumor spheroid models (MCTS).

Results. We found that stable polyplexes could be formed at physiological temperature by tuning the block lengths of the NPD polymers and the N/P ratio, reflecting the ratio between cationic nitrogen of the polymer and anionic phosphate groups in the nucleic acids, respectively. Upon encapsulation of siRNA polyplexes in the thermosensitive hydrogel, a sustained release profile was observed in vitro and thorough characterization of the released particles showed that they are stable, have the same size as before encapsulation and retain similar transfection activity. Penetration and diffusion of free siRNA polyplexes were monitored via live-cell imaging into MCTS, reaching the core of spheroids after 24 hours of incubation. A pilot in vivo study showed that fluorescently labeled polyplexes could be released locally for a least 7 days after injection intraperitoneally and subcutaneously.

Conclusions. Injectable hydrogels can enhance the local concentrations of delivered nanoparticles. We showed successful encapsulation of siRNA based polyplexes and sustained release from an injectable matrix both in vitro and in vivo. Moreover, in situ released polyplexes proved to easily penetrate hard tissue models. These formulations showed to extend the residence time of siRNA therapeutics and thereby have great potential for local therapeutic delivery.

Acknowledgment.
The Netherlands Organization for Scientific Research (NWO/VIDI 13457 and NWO/Aspasia 015.009.038) is acknowledged for funding.

Co-delivery of two genes into individual cells by means of polymeric or lipid nanocarriers is of utmost importance in many experimental settings, addressing either fundamental scientific questions or therapeutic applications. Examples are the expression of heterodimeric proteins such as antibodies or the co-introduction of an imaging marker. For widely used DNA-based gene transfer, multiple transcription units can be easily integrated on one molecule, e.g., plasmids. This strategy is not feasible for mRNA delivery. Here, co-delivery has to be achieved by mixing independent functional units or shifting the separation of functional entities to the level of proteins. In this study, we investigated multiple approaches to achieve simultaneous expression of two proteins at the same cells with a focus on formulation of nucleic acid (NA) payload, here mRNA as well as pDNA. The first strategy relies on the delivery of the genetic information via two independent “monocistronic” NAs, whereas in the second approach, this information is combined in a single NA molecule, referred here as “bicistronic”. The latter results in expression of two proteins from a single open reading frame and its co-translational separation by so called “2A” sequences. Two reporter proteins, enhanced green fluorescent protein (EGFP) and mCherry, as well as EGFP together with another non-fluorescent protein were utilized to evaluate these alternative methods, an advantageous feature of using NA as delivery entity. The data revealed that there was negligible differences between monocistronic and bicistronic delivery of genes of the same size, i.e. EGFP and mCherry. However, a significant difference between cells transfected with two genes with different molecular size was observed, in terms of cell population expressing both proteins, i.e. double positive cells. More precisely quantified by flow cytometry, 36.76±2.03% of cells were double positive for bicistronic method versus 90.43±1.27 % double positive cells, which was recorded for cells co-transfected with two monocistronic NAs. Our findings could be served as guideline for selecting the best-suited approach by choosing payload properties that can be complemented by adapting the formulation, e.g., polymer- or lipid-based, to the intended application.

Bacterial infections form part of the major causes of mortality and mobility around the world more especially in developing and under-developed countries. This study is based on the synthesis of two cationic porphyrins, 5,10,15,20-tetra-kis(4-aminophenyl)porphyrin (TAP) and 5,10,15,20-tetra-kis(4-pyridyl)porphyrin (TPyP) that are evaluated against bacterial strains by the micro-well assay and the micro-dilution method. Gold nanorods were encapsulated with each of these porphyrins and dissolved in DMSO. At 50mg/ml, the porphyrin-AuNRs produced zones of inhibition of 10-12mm against S. aurea (ATCC 25925), E. Faecalis and K. pneumonia (ATCC 4352). The minimum inhibitory concentration (MIC) values that were obtained using the micro-dilution method were, 0.78, 1.5 and 2.6mg/ml for S. aurea (ATCC 25925), E. Faecalis and K. pneumonia (ATCC 4352) respectively for all bacterial strains using TPyP@AuNRs, and 1.8, 0.42 and 0.76 mg/ml for S. aurea (ATCC 25925), E. Faecalis and K. pneumonia (ATCC 4352) respectively using TAP@AuNRs. The results show the porphyrin-AuNR conjugates to have a potential as anti-bacterial agents.

It has been fifty-four years since N-acryloyl glycinamide (NAGA) monomer and its polymer (PNAGA) were reported in 1964. It is characterized by two amides in its side chain. The concentrated aqueous solution of PNAGA has been shown to form supramolecular polymer (SP) hydrogels which are physically crosslinked by dual-amide hydrogen bonds, and the SP hydrogels’ mechanical properties can be tuned by varying initial monomer concentration, substitution groups as well as feature monomer copolymerization. Recently, our group has reported on high strength and soft PNAGA SP hydrogels by modulating hydrogen bonding density, and these SP hydrogels are developed as 3D printing bioinks for regeneration of osteochondral defect, an autolytic high strength instant adhesive hydrogel for emergency self-rescue, and a Janus hydrogel wet adhesive for internal tissue repair and anti-postoperative adhesion. The Janus hydrogel wet adhesive with strikingly distinct adhesive/nonadhesive properties on its two sides is fabricated by gradient polyelectrolyte complexation via one-sided dipping of carboxyl-containing hydrogel in cationic oligosaccharide solution. This Janus hydrogel demonstrates an instantly robust adhesion to soft tissues under water, and is successfully used for repairing perforated stomach of rabbits, meanwhile preventing post-operative tissue adhesion in vivo.

Accumulating work points out relevant genes and signaling pathways hampered in human disorders as potential candidates for therapeutics. Developing nucleic acid-based tools to manipulate gene expression, such as siRNAs, mRNA and genome editing strategies, open up opportunities for personalized medicine. Yet, although major progress was achieved in developing RNA targeted delivery carriers, mainly by utilizing monoclonal antibodies (mAbs) for targeting, their clinical translation has not occurred. In part because of massive development and production requirements and high batch-to-batch variability of current technologies, which relies on chemical conjugation. Here we present a self-assembled modular platform that enables to construct theoretically unlimited repertoire of RNA targeted carriers. The platform self-assembly is based on a membrane-anchored lipoprotein, incorporated into RNA-loaded lipid nanoparticles that interact with the antibody Fc domain. We show that a simple switch of 8 different mAbs, redirects specific uptake of siRNAs by diverse leukocyte subsets in vivo. The platform therapeutic potential is demonstrated in an inflammatory bowel disease model, by targeting colon macrophages to reduce inflammatory symptoms, and in Mantle Cell Lymphoma xenograft model, by targeting cancer cells to induce cell death and improve survival. In addition, I will discuss novel approach for delivering mRNA to specific cell types in vivo utilizing this platform. Finally, I will share new data showing high efficiency genome editing approaches in glioma and metastatic ovarian cancer. This modular delivery platform can serve as a milestone in turning precision medicine feasible.

The cellular uptake of engineered RNA, both as research tool or for (envisaged) therapeutic application came in the focus of nanomedicine by studies using short interfering RNAs (siRNA). While chemical modifications of the active ingredient siRNA proved important for efficacy of in vitro delivery, the major technological hurdles for in vivo delivery were targeting the RNA-containing nanoparticles to the right location and their in vivo delivery and survival. These bottlenecks have to be addressed by application-tailored formulations, mostly based on lipids or polymers. Part of the lessons learned can be applied for the more recent emergence of in vitro transcribed messenger RNAs (IVT-mRNA) for either vaccination of therapeutic gain-of-function approaches. Part of this spike in interest results from the safety profile (and record) of IVT-mRNA, due to its distinct chemical identity when compared to other “gene medicines”: mRNA only interferes transiently with the homeostasis of a cell’s transcriptome and does not effect the integrity of a cell’s genome composed of DNA, as has been shown for, e.g., viral delivery. Still, aside from the already mentioned hurdles in targeting and in vivo uptake, effective delivery of mRNAs requires further research in immune evasion strategies, as transfected mRNAs – among other reasons – are mistaken as viral invaders and tackled accordingly. I report on the proper use of chemically modified nucleotides as IVT-mRNA building blocks, and cell-based in vitro readout systems to monitor and ultimately modulate the innate immune response in the context of different formulations for delivery.


References:

Dammes N & Peer D (2020) Paving the Road for RNA Therapeutics, Trends in Pharmacological Sciences, 41:755

Moradian H, Roch T, Lendlein A & Gossen M (2020) mRNA Transfection-Induced Activation of Primary Human Monocytes and Macrophages: Dependence on Carrier System and Nucleotide Modification, Scienfific Reports 10:4181

Moradian H, Lendlein A & Gossen M (2020) Strategies for Simultaneous and Successive Delivery of RNA, Journal of Molecular Medicine (in press)

Recent technological advances in the production of in vitro transcribed mRNA (IVT-mRNA) facilitate its clinical use as well as application in basic research. In this regards, numerous chemical modifications, which are not observed in nature, have been introduced and successfully implemented to increase its stability, biological performance, and in particular the resulting protein production levels. Despite pronounced differences in expression levels of chemically modified IVT-mRNA as observed between different modifications, it is unclear whether these differences are exclusively due to translation capacity of chemically modified mRNAs, or if it is also caused by, e.g. differences in complex formation efficiency of modified mRNAs or in different uptake rates of IVT-mRNA/carrier complexes. Here a co-transfection experiment was designed using IVT-mRNA coding either of the two different fluorescent protein markers, i.e. EGFP and mCherry, which were synthesized with 5-methoxy-uridine (5moU) or a combination of pseudouridine and 5-methyl-cytidine (Ψ/5meC) modifications, respectively – and vice versa. We found that 5moU modified IVT-mRNA resulted in a much higher transgene expression, when transfected in human macrophages. This analysis was completed by biochemical experiments clarifying the contribution of other steps identified as possible causes for the observed differences in apparent expression levels. We predict that our findings shed light on the applicability of different mRNA modifications, and benefit future research by helping to select the proper chemical composition to accomplish their experimental/scientific requirements.

Although polycationic vectors display excellent performance in vitro with many cellular systems, their clinical use remains very restricted. To some level, this is due to the poor compatibility of such systems with biological fluids and tissues. In addition, in contrast to the processes controlling the complexation, targeting and uptake of polycationic gene delivery vectors, such as poly(ethylene imine) and poly(dimethylaminoethly methacrylate), the detailed molecular mechanisms regulating their cytoplasmic dissociation remains poorly understood. Upon cytosolic entry, gene delivery vectors become exposed to a complex, concentrated mixture of molecules and biomacromolecules. To explore cytosolic release mechanisms, we characterised the cytoplasmic interactome associated with a polycationic vector based on poly(dimethylaminoethyl methacrylate) (PDMAEMA) brushes grafted from nanoparticles. Such cationic brushes were found to be particularly effective at trapping small RNAs, resulting in high knock down efficiencies^1-3. However, how such stable association is disrupted in the cytosol was not clear. To quantify the contribution of different classes of low molar mass molecules and biomacromolecules to RNA release, we used fluorescence microscopy and developed a kinetic model based on competitive binding. We propose that the molecular structure and architecture (in particular the high surface density) of cationic brush-decorated nanoparticles, together with the cytosolic molecular crowding, modulate competitive binding and, in turn, the long term release of RNA. Based on these observations, we chemically designed polymer brushes with improved RNA retention in the cytosol, avoiding burst release, and enabling to achieve long term (at least 10 days) knock down (>70%) with one single transfection. Understanding the mechanism regulating cytosolic dissociation will enable the improved design of cationic vectors for long term gene release and therapeutic efficacy.

ACKNOWLEDGEMENTS
Funding from the ERC (ProLiCell, 772462) is gratefully acknowledged.

REFERENCES
(1) Li, D.; Sharili, A. S.; Connelly, J.; Gautrot, J. E. Highly stable RNA capture by dense cationic polymer brushes for the design of cytocompatible, serum-stable siRNA delivery vectors. Biomacromolecules 2018, 19 (2), 606.
(2) Li, D.; Wu, L.; Qu, F.; Ribadeneyra, M. C.; Tu, G.; Gautrot, J. Core-independent approach for polymer brush-functionalised nanomaterials with a fluorescent tag for RNA delivery. Chemical Communications 2019, 55 (94), 14166.
(3) Qu, F.; Li, D.; Ma, X.; Chen, F.; Gautrot, J. E. A kinetic model of oligonucleotide-brush interactions for the rational design of gene delivery vectors. Biomacromolecules 2019, 20, 2218.

The perspective of using graphene and its derivatives in bionanotechnology opens innovative and fascinating scenarios in the future of biomedicine for drug delivery, DNA sensing, protein assays, tissue engineering and cell imaging studies. For these purposes, there is an increasing interest in shedding light on the interaction mechanisms of graphene oxide (GO) live matter. In fact, the effects on human health of GO, and on cytotoxicity, are still largely unknown. Here we show that, by minimizing the oxidation degree of GO, its toxicity is significantly reduced in mouse fibroblasts. We discover a peculiar internalization of the oxidised nano graphene (nGO) leading to an intriguing nucleus decoration (ND) that could open the route to new opportunities in drug delivery. We demonstrate that it is possible to quantitatively assess the cell morphology during decoration process with nGO by a time-lapse monitoring in phase-contrast digital holography configuration. This study could open the route for further investigations on the use of label-free and quantitative imaging for characterizing the effect of graphene-based nanomaterials and their interactions with biological systems.
References
[1] Mugnano, M., Lama, G.C., Castaldo, R., Marchesano, V., Merola, F., del Giudice, D., Calabuig, A., Gentile, G., Ambrogi, V., Cerruti, P. and Memmolo, P., 2019. Cellular Uptake of Mildly Oxidized Nanographene for Drug-Delivery Applications. ACS Applied Nano Materials, 3(1), pp.428-439.
[2] Castaldo, R.; Lama, G. C.; Aprea, P.; Gentile, G.; Lavorgna, M.; Ambrogi, V.; Cerruti, P. Effect of the Oxidation Degree on SelfAssembly, Adsorption and Barrier Properties of Nano-Graphene. Microporous Mesoporous Mater. 2018, 260, 102−115.
[3] Paturzo, M.; Pagliarulo, V.; Bianco, V.; Memmolo, P.; Miccio, L.; Merola, F.; Ferraro, P. Digital Holography, a Metrological Tool for Quantitative Analysis: Trends and Future Applications. Optics and Lasers in Engineering 2018, 104, 32−47.
[4] Calabuig, A.; Mugnano, M.; Miccio, L.; Grilli, S.; Ferraro, P. Investigating Fibroblast Cells Under “Safe” and “Injurious” Blue-Light Exposure by Holographic Microscopy. Journal of biophotonics 2017, 10, 919−927.

An increasing number of studies report that exosomes play critical roles in intercellular communication. While exosomes are essential to maintain physiological conditions, aberrant exosomal communication can lead to the development of cancer, diabetes, and many other diseases. Here we will describe new materials to redirect and modulate cellular communication mediated by exosomes for therapeutic applications. Specifically, we have identified RNA-based materials to modulate exosomal function by repackaging them with different types of cargoes. This presentation will discuss how beneficial exosomal communication can be enhanced for therapeutic purposes.

Metal-organic frameworks (MOFs) have emerged as promising functional biomaterials for a wide range of applications in drug delivery and biomedical imaging. In particular, nanoscale MOFS (nMOFs) have drawn significant attention as novel therapeutic agents in oncology both as drug carriers and as stimuli-responsive materials. Here we present our recent findings utilizing nMOFs as a multipronged radiosensitizer for chemoradiation in breast and colorectal cancer models. Through incorporation of high-Z elements, such as Hf, we demonstrated an enhanced physical radiation dose deposition observed though increased production of reactive oxygen species generation and subsequent DNA double strand breaks in X-ray irradiated cells. Coupled with delivery of DNA repair inhibitors, such as talazoparib (PARPi), we found improved control of tumor growth when employing nMOFs with radiation therapy (RT). We further investigated the integration of functional coatings, including fucoidan targeting P-selectin expression, to improve delivery of these nanomaterials during fractionated RT. In addition, we describe here the interaction of X-rays with lanthanide-based nMOFs to serve as energy mediators in deep tissue photodynamic therapy (PDT) or molecular probes in radioluminescence imaging. Detailed physiochemical characterization and in vivo toxicity data will be presented to promote discussion on clinical translation challenges of these novel materials.

Sustained and localized drug delivery in the oral cavity remains an important challenge in pharmaceutical sciences.¹ Currently, existing self-applied dosage forms such as gels, toothpaste or mouthrinses are rapidly washed away after administration, resulting in modest efficacy and thus require repeated applications. Therefore, oral devices that fit precisely patient’s anatomy² with spatiotemporally controlled drug release profiles represent a promising approach to improve drug delivery in the oral cavity. Here, we demonstrate how the 3D printing of drug-polymer composite materials could be utilized to achieve this goal. Thanks to the possibility of printing complex 3D geometries with multiple materials³, the fused deposition modelling (FDM) technique was chosen to fabricate personalized mouthguards that allow local and prolonged elution of drugs.
As a model drug compound, sodium fluoride (NaF), a salt commonly used to prevent tooth decay, was combined with a variety of polymer mixtures containing different ratios of poly(ε-caprolactone), poly(ethylene glycol) and poly(vinyl alcohol). Composite filaments with tunable hydrophobicity were then produced from the blends of thermoplastics with varying loads of NaF and fed into the FDM printer. The mechanical properties were assessed with standard tensile testing to confirm the suitable toughness and durability of the printed materials. In vitro dissolution studies further demonstrated the tunable release kinetics of the devices.
Finally, toothguard models that fit precisely the geometries of human teeth were designed after 3D scanning of the specimens and were then printed in high resolution. To evaluate the therapeutic effects of the devices, the toothguards were used ex-vivo on the decayed teeth for 7 days. The lesion was characterized by scanning electron microscopy and the fluoride incorporation was measured by energy-dispersive X-ray spectroscopy. A significant elevation of fluoride contents in the enamel was found in the specimen in contact with the fluoride-loaded devices compared with the one in contact with simple control solutions. In conclusion, the data show that a slow and local drug release of fluoride could improve the buccal delivery of therapeutic compounds.

Nano-sized materials have the unique capacity to distribute in organisms and enter cells easily using cellular pathways. This has opened up tremendous opportunity in nanomedicine for the use of nano-sized carriers to deliver drugs more efficiently to their site of action. However, the molecular details of the mechanisms of uptake and intracellular trafficking of nano-sized materials are in most cases still not clear. Such knowledge could allow us to further improve the design of truly targeted nanomedicines.
To this aim, we have combined the use of transport inhibitors and RNA interference with proteomic based methods to identify the cell surface receptor interacting with nano-sized materials and characterize the mechanisms by which they are internalized by cells.^1,2 Additional efforts have been focused on developing in vitro endothelial cell barriers more closely resembling the barriers nanomedicines encounter in vivo.³ Uptake and distribution of nanoparticles have also been studied in precision cut tissue slices from the major organs in which nanoparticles distribute, such as the liver, lungs and kidneys, as an advanced ex vivo 3D model.^4,5
Our results show that the same cells process nanoparticles in different ways when they are developed into an endothelial cell barrier rather than at different degrees of cell density, as commonly applied for in vitro studies.³ Furthermore we show that liver tissue slices allow to resemble ex vivo some of the major outcomes of in vivo distribution studies, i.e. the preferential accumulation of nanoparticles by Kupffer cells, the liver macrophages.⁴ Finally, we show that the corona molecules adsorbing on the nanoparticle surface once applied in serum or plasma can be recognized by specific cell receptors, and they can also affect the details of the following uptake mechanism by cells.⁶ Thus, in other words, the same nanoparticles enter cells via different pathways when different coronas are formed on their surface. Using liposomes of different composition, we also show how the composition of the corona forming in serum can be tuned, thus affecting uptake efficiency and uptake kinetics.⁷

References:

¹ Comparison of the uptake mechanisms of zwitterionic and negatively charged liposomes by HeLa cells, D Montizaan, et al, Nanomedicine NBM, 2020, 30, 102300
² Limits and challenges in using transport inhibitors to characterize how nano-sized drug carriers enter cells, V Francia, et al, Nanomedicine 2019, 14 (12), 1533-1549
³ Effect of the development of a cell barrier on nanoparticle uptake in endothelial cells
V Francia, et al, Nanoscale 2018 10 (35), 16645-16656
⁴ Time-Resolved Quantification of Nanoparticle Uptake, Distribution, and Impact in Precision-Cut Liver Slices, R Bartucci, et al, Small, 2020, 1906523
⁵ Comparative study of nanoparticle uptake and impact in murine lung, liver and kidney tissue slices
R Bartucci, et al, Nanotoxicology, 2020, 14 (6), 847-865
⁶ Corona Composition Can Affect the Mechanisms Cells Use to Internalize Nanoparticles
V Francia, et al, ACS nano 2019 13 (10), 11107-11121
⁷ Tuning Liposome Composition to Modulate the Corona Forming in Human Serum and Uptake by Cells, K Yang, et al, Acta Biomaterialia, 2020, 106, 314-327

Chitosan is an abundantly common, naturally occurring, polysaccharide biopolymer. Its biocompatible, biodegradable, and antimicrobial properties have led to significant research toward biological applications such as drug delivery, artificial tissue scaffolds for functional tissue engineering, and wound-healing dressings. For applications such as tissue scaffolding, formation of highly porous mats of nanometer-sized fibers, such as those fabricated via electrospinning demonstrated high potentials.

In this framework, we have as first objective to control better the scaffold porosity by decreasing the fiber density while keeping enough mechanical performances to be handled and implanted. By playing on the design of the collector used for electrospinning, we have developed structured fiber mats that meet both targeted high porosity and mechanical resistance properties.

In a second objective, strategies have been developed allowing proteins encapsulation within the chitosan scaffolds for locoregional release. The electrospinning process has been optimized in order to preserve the protein integrity and its biological activity. Various release profiles of the protein have been achieved from the structured electrospun scaffolds. A sustained release has been obtained by the use of proteins loaded poly(lactic-co-glycolic acid) (PLGA) nanoparticles [1].

The developed electrospinning process allowing both the precise control of the porosity and of the release profile of a protein will be useful for elaborating porosity and concentration gradients which are critical when developing biomimetic scaffolds aiming to control cell migration and trapping.

[1] Haji Mansor M, Najberg M, Contini A, Alvarez-Lorenzo C, Garcion E, Jérôme C & Boury F. Development of a non-toxic and non-denaturing formulation process for encapsulation of SDF-1α into PLGA/PEG-PLGA nanoparticles to achieve sustained release. European Journal of Pharmaceutics and Biopharmaceutics. 2018; 125: 38-50

Daily drug regimen adherence is a widespread challenge for individuals living in all countries. For those in low and middle income countries (LMICs), there is an added layer of complexity due to a number of systemic factors, including poor access to medications in rural areas in combination with a shortage of healthcare workers and facilities, weak supply chains, and the challenge of “last mile” transportation. The lack of infrastructure in LMICs presents further challenges for product development. Cold chain is sporadic and often absent, and electricity often is not reliable or available. Hence, there is a need for products that provide long-term shelf stability at ambient temperatures. The Gates Foundation has built a broad portfolio of long-acting drug delivery technologies that address these several of these issues and have the potential to increase uptake of essential medicines. These investments span a spectrum of materials science approaches and support a diverse array of use cases in HIV prevention and family planning contraception.

Polymeric electrospun nanofibers have extensive applications in filtration, sensing, drug delivery, and tissue engineering that often require the fibers to be patterned or integrated with a larger device. Here, we describe a highly versatile in situ strategy for three-dimensional electrospun fiber patterning using collectors with an insulative surface layer and conductive recessed patterns. We show that two-layer
collectors with pattern dimensions down to 100-micrometers are easily fabricated using available laboratory equipment. We use finite element method simulation and experimental validation to demonstrate that the fiber patterning strategy is effective for a variety of pattern dimensions and fiber materials. Finally, the potential for this strategy to enable new applications of electrospun fibers is demonstrated by incorporating electrospun fibers into dissolving microneedles for the first time. These studies provide a framework for the adaptation of this fiber patterning strategy to many different applications of electrospun fibers.

Hydrogels, water-swollen cross-linked polymeric networks, resemble natural soft tissues (e.g. skins) more than any other types of materials including silicones. They are representative soft biomedical materials in terms of a high water content over 70%, good biocompatibility, low foreign body sensation, and favorable capacity of water-soluble molecules. In this regard, hydrogels are promising materials for drug delivery system as the form of patches, contact lens, and synthetic skins. As a platform for drug delivery, simultaneously accomplishing the fast delivery of a drug and shape conformity of a hydrogel can find its use of pharmaceutical or cosmetic patches. However, there exists the trade-off between the liquid and solid properties that the liquid medium like eye-drops rapidly releases a drug but cannot be used as a patch on human skins, whereas a solid-state hydrogel realizes the stable drug delivery platform but inevitably exhibits a slow release of the drugs.
Herein, a malleable and biocompatible hydrogel containing micellar cargo-expellers was prepared. The in situ polymerization of poly(hydroxyethyl methacrylate) (PHEMA) within the poly(vinyl alcohol) (PVA)-borax hydrogel matrix produces large compound micelle particles (micelles) that are not bound by the matrix. Notably, as the micelles do not form any chemical crosslinks with the matrix, they rapidly release outside of the hydrogel network in a wet condition. The micelles act as cargo-expellers through a concentration gradient, which delivers a 25-fold larger quantity of a model cargo than the control hydrogel. Also, the dynamic borate-diol bonds of the hydrogel matrix engender the hydrogel with self-healing properties, and mechanical shock-absorbing property of micelles enables the hydrogel to tightly contact highly curved skin. Moreover, biocompatibility of the hydrogel was confirmed by both in vivo cytotoxicity and in vivo skin irritation tests. Consequently, this malleable hydrogel will inspire novel prompt skin-patch systems for pharmaceutical and cosmetic purposes. [1]

[1] S.-H. Shin et al. Adv. Healthc. Mater. 2020, 9(19), 2000876.

Current approaches for drug delivery to solid tumors like glioblastoma are insufficient. Delivery of passive- and active-targeting nanomaterials is diffusion-limited, hindered by heterogenous tumor vasculature, and has resulted in few improvements to the standard of care for rapid and lethal disease. Macrophages readily survey solid tumors and are actively involved at all stages of tumor development. Here we introduce genetically engineered macrophages (GEMs) with a fully bioorthogonal, genetically encoded receptor capable of loading with polymeric prodrugs (referred to as ‘drugamers’) in a titratable manner. These RAFT-based polymers are multi-functional: affording biocompatibility, solubility, and sustained release of a small molecule drug cargo. Using the PI3K inhibitor, PI-103, as a model drug, we demonstrate how our polymeric formulation’s optimized release kinetics compliment the drug’s mechanism of action, leading to improved and sustained activity against cancer cells. Furthermore, our drugamer-loaded GEM platform introduces a new cell-mediated and kinetically regulated mechanism for delivery of small molecule drugs to tumors. We have previously demonstrated that GEMs home to and persist in tumors following both intratumoral and intravenous delivery, suggesting the potential of this cellular therapeutic to be administered intravenously to localize to single sites or even metastases.

Endosomal sequestration of lipid-based nanoparticles (LNPs) remains a formidable barrier to intracellular delivery of mRNA. Herein, structure-activity analysis of cholesterol analogues reveals that incorporation of C-24 alkyl phytosterols into LNPs (eLNPs) enhances gene transfection and the length of alkyl tail, flexibility of sterol ring and polarity due to -OH group is required to maintain high transfection. Cryo-TEM displays a polyhedral shape for eLNPs compared to spherical LNPs, while x-ray scattering shows little disparity in internal structure. eLNPs exhibit higher cellular uptake and retention, potentially leading to a steady release from the endosomes over time. 3D single-particle tracking shows enhanced intracellular diffusivity of eLNPs relative to LNPs, suggesting eLNP traffic to productive pathways for escape. Our findings show the importance of cholesterol in subcellular transport of LNPs carrying mRNA and emphasize the need for greater insights into surface composition and structural properties of nanoparticles, and their subcellular interactions which enable designs to boost intracellular delivery of mRNA. I will briefly discuss three applications of LNPs to deliver mRNA i.e. for the treatment of cystic fibrosis, retinal degeneration and COVID-19 therapeutics.