Symposium S.SM08—Emerging Strategies and Applications in Drug Delivery

The reactive oxygen species (ROS)-mediated mechanism is the major cause underlying the efficacy of photodynamic therapy (PDT). The PDT procedure is based on the cascade of synergistic effects between light, a photosensitizer (PS) and oxygen, which greatly favors the spatiotemporal control of the treatment. This procedure has also evoked several unresolved challenges at different levels including (i) the limited penetration depth of light, which restricts traditional PDT to superficial tumors; (ii) oxygen reliance does not allow PDT treatment of hypoxic tumors; (iii) light can complicate the phototherapeutic outcomes because of the concurrent heat generation; (iv) specific delivery of PSs to sub-cellular organelles for exerting effective toxicity remains an issue; and (v) side effects from undesirable white-light activation and self-catalyzation of traditional PSs. In this talk, the current status and the possible opportunities of nanomedicine for ROS generation for cancer therapy will be discussed in detail.

The central nervous system (CNS) plays a central role in the control of sensory and motor functions, and the disruption of its barriers can result in severe and debilitating neurological disorders. Neurotrophins are promising therapeutic agents for neural regeneration in the damaged CNS. However, their penetration across the blood-brain barrier remains a formidable challenge, representing a bottleneck for brain and spinal cord therapy. We have developed a nanocapsule-based delivery system that enables intravenously injected nerve growth factor (NGF) to enter the CNS in healthy mice and nonhuman primate. In pathological conditions, the delivery of NGF enables neural regeneration, tissue remodeling, and functional recovery in mice with spinal cord injury. This technology can be utilized to deliver other neurotrophins and growth factors to the CNS, opening a new avenue for tissue engineering and the treatment of CNS disorders and neurodegenerative diseases.

Introduction: Cancer is the second leading cause of death in the world and current treatment methods have low 5-year survival rates. One potential solution to improving treatment methods is targeted nanoparticle drug delivery. Nanoparticles can be engineered to increase the efficacy of chemotherapeutic drugs through increased targeting and dose delivery. Despite proven benefits of nanoparticle drug delivery, it comes with its own challenges, the largest of which is overcoming the physiological barriers between the nanoparticle injection site and the tumor in the body. In order to better understand the pharmacokinetics of nanoparticles in vivo, nanoparticle characteristics need to be varied in a systematic study to identify ideal formulations. This systematic study would need to test nanoparticles of different sizes, shapes, and chemistries with multiple phases, and these properties would need to be independently varied. Current fabrication methods are unable to make monodisperse, multiphasic or non-spherical particles and spherical particles, and to independently alter each parameter.

Objective: To address the need of a nanoparticle fabrication method which can modulate particle properties in a systematic and independent manner, we have developed a novel polymeric particle synthesis technique, called Wettability Engendered Templated Self-assembly (WETS) and have demonstrated the ability to control size, shape and composition independently.

Methods and Results: The WETS methodology uses surfaces with patterned wettability to self-assemble discrete, monodisperse polymer particles in an array after dip-coating in a polymer solution. These particles can have dimensions ranging from 25 nm to 150 µm, and can have a variety of non-spherical, planar geometries such as disks, squares, triangles and hexagons. Size and shape are independently controlled through the patterned wettability surface and the dip-coating parameters. Additional dip-coating layers of other polymers on top of previously deposited polymer layers allows for the fabrication of multiphasic particles with each phase able to independently encapsulate a different therapeutic or diagnostic agent. As each phase is added to the particle individually, the size and composition of the phases can be independently altered. Single and multiphasic spherical particles are formed from the reconfiguration of non-spherical particles in a predictable manner. Multiphasic spherical particles can be tuned to create Janus or core-shell morphologies based on reconfiguration parameters. These results show the ability to synthesize varied particles with a single method which is required to make direct comparisons between particle characteristics in order to develop improvements in cancer therapies.

Conclusion: With the novel WETS fabrication method, we have demonstrated the ability to have systematic and independent control over the size, shape and composition of polymeric particles in a predictable manner. This method will allow for a systematic study of nanoparticle pharmacokinetics in vivo in order to identify ideal nanoparticle formulations for cancer therapies. Continued work is focused on the scale up of WETS in order to undertake in vitro and in vivo studies in the future.

Magnetic nanoparticles under the influence of the external magnetic field have been widely studied for the past few decades due to their potential applications in bio and nanotechnology. However, the interplay of dipolar and Zeeman interactions during the assembly process is a major question that remained to be answered. Current modeling and simulation techniques are primarily limited to coarse-grained methods, which lack proper resolution for understanding these processes at the atomic level. In this work, we developed a novel method to study the dipolar and Zeeman interactions within stable single domain (SSD) magnetic nanoparticles using atomistic molecular dynamics simulation. The primary advantage of this technique is the atomistic resolution that permits the study of magnetic nanoparticles' alignment and their explicit interactions with the surrounding environment. Our methodology permits the investigation of the formation of chain and ring shapes of 3.2 nm and 8.2 nm cubic and spherical Magnetite (Fe3O4) nanoparticles functionalized with Oleic Acid under the influence of an external magnetic field and detailed investigation of the role of solvent properties on shape formation. This developed method can be used as a plugin with the LAMMPS molecular dynamics software to study the behavior of small magnetic nanoparticles to gain insights into the physics and chemistry of different magnetic assembly processes.

Enabled initially by the development of microelectromechanical systems, current microfluidic pumps still require advanced microfabrication techniques to create a variety of fluid-driving mechanisms. Here we report a generation of micropumps that involve no moving parts and microstructures. This micropump is based on a principle of photoacoustic laser streaming and is simply made of an Au-implanted plasmonic quartz plate. Under a pulsed laser excitation, any point on the plate can generate a directional long-lasting ultrasound wave which drives the fluid via acoustic streaming. Manipulating and programming laser beams can easily create a single pump, a moving pump, and multiple pumps. The underlying pumping mechanism of photoacoustic streaming is verified by high-speed imaging of the fluid motion after a single laser pulse. As many light-absorbing materials have been identified for efficient photoacoustic generation, photoacoustic micropumps can have diversity in their implementation. These laser-driven fabrication-free micropumps open up a generation of pumping technology and opportunities for easy integration and versatile microfluidic applications

In this talk, we will present our recent work on the synthesis and engineering of supramolecular hydrogels for controlled drug delivery and thermal stabilization of encapsulated biologics. Polymer–nanoparticle (PNP) hydrogels have been produced from drug-loaded nanoparticles (NPs) and cross-linking polymers. These transient physical assemblies are formed through supramolecular interactions between the NPs and the polymers and exhibit shear-thinning and self-healing behavior suitable for injection or extrusion. In our lab, we are applying PNP hydrogels for local injection of therapeutics and as immune-modulatory biomaterials. In addition, we have developed the platform as a universal rheological carrier for biomaterials additive manufacturing. Uniquely, the nano-carrier can accommodate a range of secondary polymers for post-print stabilization and functionality. We are exploiting these printable PNP gels to assemble additively manufactured drug delivery systems for dual release of both hydrophilic and hydrophobic molecules. We also employ dynamic covalent chemistry to design moldable polymeric hydrogels. Here, we have fabricated ideal reversible networks and investigated how the molecular characteristics of the dynamic covalent cross-links impact the emergent material properties. We have leveraged this understanding to design biomaterials that can encapsulate, thermally stabilize, and release temperature-sensitive biologics. We are exploiting these materials to mitigate the global reliance on the cold chain in the distribution of value-added biologics and biotherapeutics, including vaccines. In total, this talk will illustrate how molecular design of hydrogels can enable emerging applications in drug delivery from injectable biomaterials, additvely manufacturing controlled release systems, and materials for the thermal stabilization of biologics.

In biological fluids, proteins are adsorbed onto the surface of nanoparticles (NPs) to form a coating known as protein corona. Most of the corona proteins act as opsonin which activates the macrophage from immune system to uptake NPs, leading to the rapid removal of NPs [1]. This restricts the development of nanomedicine. Although conjugation with linear polyethylene glycol (PEG) is the standard approach to reduce protein attachment and to avoid non-specific uptake, it cannot fully prevent the opsonization. On the other hand, we have demonstrated polyglycerol (PG) as a promising alternative to PEG, because PG enhanced the aqueous dispersibility and gave stealth effect to NPs [2]. In order to understand the role of PG, we compare protein affinity and stealth effect of PG and PEG grafted nanodiamond (ND-PG and ND-PEG, respectively) with different density in this paper. Protein analyses by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) indicated that PG was much more resistant than PEG to adsorption of the opsonin proteins such as IgG and complement protein. In particular, there was almost no protein on the dense PG layer. In vitro stealth effect was revealed by TEM; almost no ND-PG was observed in the TEM images of U937 macrophage, while there was ND-PEG in the macrophage. This indicates that PG has much better stealth effect than PEG. In vivo stealth effects including blood circulation and biodistribution will be reported in due course.
References: 1) S. Schöttler et al., Nat. Nanotechnol., 2016; 2) L. Zhao, N. Komatsu et al., Angew. Chem. Int. Ed. 2011

High density lipoprotein (HDL) is a nanoparticle (∼8-10 nm) composed of a lipid membrane bilayer wrapped around by a “belt” of amphipathic helices of Apolipoprotein A-I (ApoA-I). HDL travels in directly interacts with macrophages in arterial plaques to efflux excess cholesterol and subsequently deliver it to the liver by a process of reverse cholesterol transport (RCT). Our research focus is on design of synthetic high density lipoprotein (HDL) nanoparticles for treatment of cardiovascular disease. We discover novel ApoA-I mimetic peptides, explore biophysics of peptide-lipid interactions and optimize HDL compositions for interaction with specific cellular receptors, transporters and enzymes involved in RCT. We examine sHDL biodistribution, pharmacokinetics and pharmacodynamic properties. We take advantage of small size and clinical safety of sHDL to use is it platform for drugs and vaccines delivery. This talk will cover the use of sHDL nanoparticles to deliver liver X receptor agonist to atheroma to reduce inflammation and atherosclerosis. This strategy utilizes pharmacological ability of sHDL to serve as an acceptor of cholesterol effluxed from atheroma. The use of sHDL nanoparticles for personalized vaccine and chemo-immuno therapy delivery results in development lasting protective immunity against tumor reoccurrence in animal models of colon cancer, glioblastoma and melanoma.

The ability to track drug carriers in their administered environment in real time and to release the drug in a controlled, minimally invasive manner are hallmarks of an advanced drug delivery system. This presentation focuses on ultrasound-sensitive multilayer capsules as efficient contrast-enhanced imaging agents utilizing ultrasound, magnetic resonance imaging (MRI), and positron emission tomography (PET) imaging modalities. These capsules are composed of hydrogen-bonded polymers and capable of delivering biological and synthetic molecules upon low-power (~100 mW/cm²) diagnostic or high-power (>10 W/cm²) therapeutic ultrasound irradiation. We will also discuss the capsule efficacy in modulating the redox state that can influence immune responses for prolong circulation in the blood. The ability of this material to conjugate metalloporphyrin to further enhance immunomodulatory potential by dissipation of free radicals will be also presented. Owing to the active contrast, long circulation, customizable size, shape, composition, and precise delivery of high payload concentrations, these materials present a powerful and safe platform for imaging-guided precision drug delivery.

Suspension cells, which are of great interest in immunotherapy for infectious disease and cancer, remain challenging to transfer cargo into efficiently using existing nonviral delivery platforms due to either low efficiency or high death rate. Recent studies deploying the photothermal effect for intracellular delivery have shown promising results. Although researchers successfully demonstrated diverse cargo delivery into adherent cells, current platforms are not ideal from delivery into suspension cells. we demonstrate a high-efficiency photothermal delivery approach for suspension cells using nanoscale metal-coated tips at the edge of microwells. A uniform microwell array with three dimensional nanoscale metallic sharp tips is generated by a self-aligned microfabrication process. Suspension cells self-position by gravity into microwells with each cell in direct contact with 8 sharp tips. The precision control of number and locations of pores opened on cell membrane is the key to achieving high cell viability delivery using photothermal approaches. Cargo sizes ranging from 0.6 to 2,000 kDa were tested on this platform using Ramos B cells, a mature human B lymphocyte cell line. We achieve the efficiency >84% for Calcein green (0.6 kDa), >45% for large molecules FITC-dextran (2,000 kDa), active enzyme protein, and green protein fluorescence (GFP) encoded plasmids into suspension cells with their functions maintained, retained viability >96%, and a throughput >100,000 cells delivered per minute.

Metallic nanomaterials in polymers have been used in a variety of biomedical applications including drug delivery, enhanced antibacterial activity, and tumor ablation. The plasmonic response of metallic nanomaterials involves electrons that oscillate at resonance with incident light. The plasmon resonance frequency at the particles’ surface is dependent on factors such as nanoparticle (NP) size, shape, composition, state of aggregation, and environment. In the case of polymers impregnated with metallic nanomaterials, non-propagating surface plasmon modes within the electromagnetic spectrum enable the conversion of optical energy to thermal energy with a coincident temperature change within the polymer. In previous work, investigators have used polymer capsules containing metallic nanomaterials in the shell to melt the shell and release encapsulated drugs in the presence of light. Such capsules are functionalized to target cancer tissues and, once at the correct locale, are activated by light to treat tumors or other cancerous matter. For this work, we will focus on topical, electrospun fiber drug delivery systems that can be stored and activated in the presence of light to release treatments such as antibiotics or coagulants when needed.
In our previous work, colloidal gold NPs (GNPs, max. absorption λ = 522 nm) were incorporated into polymer films and electrospun into fibers to utilize the NP plasmonic response for localized heating of the polymer. Mathematical modeling was used to describe the GNP distribution and heat/melt profile surrounding each GNP under illumination in the polymer, demonstrating that a bulk temperature change of only 0.2 °C results in a 20-nm-diameter melted polymer sphere around the GNP. Such results provided us with a mathematical guide to predict the GNP size that would result in a desired melt thickness. In our previous work, it was shown that by reducing the radius of polymer material around the GNP though the use of nanoscale-diameter electrospun fibers in place of an infinite thin film (centimeter scale) heating of the bulk material increased by 72 %. This result is attributed to the reduction of polymer material surrounding each GNP. Bulk heating of polymer blend films containing GNPs were mapped using a forward-looking infrared (FLIR) camera system with light-emitting diodes (LEDs). Change in temperature observed in the thin films was related to photothermal energy conversion efficiency of the thin films through heat transfer calculations. Significantly, the GNP-doped film photothermal conversion efficiency increased by 1.8 times (75.7 % increase) under 530-nm LED illumination.
Based on the mathematical model developed, the enhanced heating observed, and the enhanced efficiencies of the materials explored thus far, future objectives include developing these materials further for light activated drug delivery systems. Future work will include developing the mathematical model for silver nanoplates (SNPs), which demonstrated a stronger absorption response and are more economical than gold. SNPs used will be doped into polymer thin films and electrospun into fibers, and bulk heating will be monitored on the FLIR system. Heating in the thin films versus nanoscale-diameter fibers will be compared, and efficiencies of the materials will be calculated. In addition, ideal photothermal heating will be studied through altered nanomaterial concentrations in the polymers. The relationship between photothermal heating and SNP concentration will elucidate any plasmonic coupling and guide material optimization schemes.

Layered polymer systems than can be fabricated using roll-to-roll methods have been demonstrated using film extrusion methods. Together with lithography, they enable the fabrication of capsules and colloidal particles that have controlled kinetics for release of agents both pre-loaded and post-loaded. In this talk, we demonstrate the scalability of drug release using a hierarchical configuration of layers that can be fabricate layer-by-layer as applied to patches, capsules, and nanosheets. The methods make use of solution methods as well as polymer multilayer extrusion methods more popularly known as the CLIPS process. The solution methods take advantage of the physisorption of polyelectrolytes to form suspendable ultrathin nanosheets. It is possible to achieve a production scale of roll-to-roll films using the CLIPS process. The controlled release of model drugs is demonstrated under buffer conditions including the demonstration on the preparation of patches and nanosheets.

Understanding the complex neural circuity and its correlation to specific behaviors requires spatially and temporally precise modulation of neuron subtypes in certain brain regions. One major challenge of existing neural modulation techniques is the tradeoff between invasiveness and depth: invasive implants of electrodes and optical fibers are usually required for electrical and optogenetic modulation of neural activity in deep brain, while non-invasive brain stimulation methods lack penetration depth and neuron-type specificity of brain modulation. The invasive brain implant and head tethering result in acute tissue damage and chronic gliosis at the neural interface, leading to degradation of recording and stimulation capabilities over time. In this talk, I will present the latest advances of minimally invasive approaches for in vivo neuron-type specific brain modulation from my lab at Stanford University. First, I will describe a new approach termed ‘sono-optogenetics’, which uses a circulation delivered rechargeable light source for non-invasive optogenetics in deep brain regions through the intact scalp and skull. In this approach, we address the challenge of limited tissue penetration of visible photons via the intravenous delivery of mechanoluminescent nanoparticles, which can act as local light sources in the brain when triggered by brain-penetrant focused ultrasound (FUS). After delivery, these particles can be recharged by 400-nm photoexcitation light in superficial blood vessels during circulation, and turned on by FUS to emit 470-nm light repetitively in the intact brain for optogenetic stimulation. Second, I will describe a type of new deep-brain stimulating devices termed ‘injectable photovoltaics’ for a wireless, gliosis-free neural stimulation interface. Micron-sized, ultraflexible and wirelessly powered microdevices can be delivered into the brain via syringe injection so as to disturb the brain as little as possible when deployed. These injectable devices are fabricated with photovoltaic polymers, turning brain-penetrating near-infrared light into electrical impulses for deep-brain neural stimulation. These two brain-modulation approaches have the potential of generating transformative results for both neuroscience research and neurological applications, not only expanding the toolbox of neuromodulation techniques, but also offering strategies for clinical translation of therapeutic neural implants.

Cas9 based therapeutics have the potential to revolutionize the treatment of genetic diseases. However, safe and effective methods for delivering the Cas9-guide RNA complex (Cas9 RNP) need to be developed before the clinical potential of Cas9 based therapeutics can be fully realized. In this presentation, I will describe non-viral delivery strategies developed in our laboratory for delivering the Cas9 RNP. The first delivery strategy is termed CRISPR-Gold. CRISPR-Gold is composed of gold nanoparticles assembled with the Cas9/gRNA ribonucleoprotein (RNP) complex, donor DNA, and an endosomal disruptive polymer. CRISPR-Gold was able to correct the DNA mutation that causes Duchenne muscular dystrophy (DMD) in mdx mice via homology directed DNA repair (HDR), with an efficiency of 5.4% after an intramuscular injection. In addition, CRISPR-Gold was able to edit the brains of adult mice and rescued mice from the repetitive behaviors caused by autism. In this presentation, I will also describe other strategies for delivering the Cas9 RNP based on encapsulation in block copolymers and conjugation with peptides. Collectively, our experience suggests that non-viral strategies for delivering the Cas9 RNP have great potential for treating DMD and other genetic diseases.

Our research combines immunology and biomaterials to understand the interactions between synthetic materials and immune tissues, and to design more selective therapeutic vaccines for cancer and autoimmunity. This presentation will highlight our recent efforts toward these goals combining materials science and bioengineering tools, cell culture, animal models, and samples from human patients. In one example I will discuss new degradable polymer depots that could improve the selectivity of therapies for autoimmune diseases such as multiple sclerosis and diabetes by locally reprogramming the function of lymph nodes – tissues that coordinate immune function. A second area will present the lab's efforts to self-assemble immune signals into modular nanostructures. This rational design approach allows activation of programmable combinations and levels of immune pathways triggered. Modular control over these aspects of immune signaling could help improve the efficacy of vaccines for cancer and infectious disease, and enhance the efficiency of vaccine translation.

Polycaprolactone (PCL) have been used in medical applications due to its inertness, biocompatibility, biosorbability and biodegradability. However, its hydrophobic nature and absence of fluid absorption capacity limits its potential in such applications. This can be improved by incorporating particles that will facilitate interaction with water or body fluids. In this study, alginate-chitosan PECs are synthesized and incorporated in PCL nanofibrous membrane. Given this structure, alginate nanoparticles were synthesized to allow incorporation into the membrane. Varying mass ratio of alginate to calcium ions were investigated in the pre-gel formation which was controlled through viscosity measurements. Viscosity decreased significantly from 1.873 to 1.515 cPs indicated the formation of the alginate particles as a consequence to the rearrangement of the long alginate polymer chains into discrete coiled structure. Meanwhile, varying mass ratio of alginate to chitosan was investigated in the polyelectrolyte complexation to facilitate the formation of a stable suspension thereby suggesting formation of alginate-chitosan nanoparticles. A minimum particle size of 1.924 microns is measured for stable solutions. The alginate-chitosan was incorporated into the PCL nanofiber membrane to impart hydrophilicity and water absorbability. Infrared spectroscopy done on the nanofiber membrane confirmed the presence of both PCL and alginate in the final structure. Further, contact angle measurements showed a significant decrease from 82.56 to 73.58° which suggests the improvement of hydrophilicity of the membrane. Water absorption capacity of the membrane increased to 7.82%. Varying the amount of alginate-chitosan particles would allow improvement and control of the nanofiber membrane’s hydrophilicty and water absorption capacity. This material has potential use in wound dressing devices, extracellular membranes, as well as filter membranes in medical devices.

Hydrogels are water-compatible polymer networks which can increase their volume without losing their shape. The applications for this type of materials depend on their maximum swelling grade and mechanical properties, which are closely related with the cross-linking porosity and the density of the network. Their flexibility degree is very similar to that of natural tissues, so the hydrogels are considered a very important group in the pharmaceutical industry for biomedical applications, particularly, as controlled drug delivery systems. The advantages of these systems include the possibility of drug delivery at specific sites, the continuity of the treatment during the night, the stability of the drug, its optimal absorption by the tissues, as well as the reduction of side effects. In this study, we report the evaluation of hydrogels of polyacrylamide (PAAm)/starch, synthesized in different molar ratios (100/0, 90/10, 80/20, 70/30, 60/40, 50/50), as a potential platform for the controlled release of amoxicillin. The swelling capacity of the hydrogels and the effect of the molar ratio between both polymers on the antibiotic release kinetics under normal physiological conditions of temperature and pH were evaluated in vitro. Finally, bacterial growth inhibition assays in ATCC strains were carried out to analyze the clinical potential of materials. The results have demonstrated the potential of polymeric hydrogels as controlled release system of amoxicillin.

Selenium (Se) ion release from a bone cement is hypothesized to provide chemotherapeutic effects and to be beneficial in the prevention of recurrence of osteosarcoma in the setting of tumor resection surgery. Se containing compounds have been shown to act as redox modulators with higher selectivity and sensitivity in malignant cells. A four glass series, SiO₂-Na₂O-CaO-SrO-SeO₂, was investigated substituting varying levels of CaO for SeO₂. The glass series was synthesized using the air-quench method. This method was repeated twice for each glass in the series in order to ensure homogeneity of the material. This study focuses on the characterization of the bioactive glass and its ability to release beneficial ions in an aqueous environment. Structural and thermal properties were evaluated for the glass series using differential thermal analysis (DTA) along with magic angle spinning nuclear magnetic resonance (MAS-NMR) to confirm network connectivity (NC) calculations. X-ray diffraction (XRD) was used to confirm the presence of an amorphous structure in the series. Inductively coupled plasma mass spectroscopy (ICP-MS) was performed in order to quantify the ion release from the glass in an aqueous environment at physiological temperature for all elements present in the glass. Antibacterial efficacy tests were completed along with cytocompatibility studies via MTT assay of the material plated conditioned media with MC3T3 cells. The glass composition was determined to have bioactive properties, detectable and predictable Se ion release, antibacterial abilities, and also promoted growth of pre-osteoblast cells. Future directions include assessing the ability of the selenium based bioglass to stop or reverse growth of osteosarcoma cells.

Dental pulp, a highly vascularized tissue situated in an inextensible environment surrounded by rigid dentinal walls, receives blood supply solely from a small apical foramen (diameter < 1 mm) of the tooth root canal. Therefore, regeneration of pulp in the full-length tooth root has long been a challenge. In this study, we aimed to develop angiogenic human dental pulp stem cells (hDPSCs) for pulp regeneration in a full-length tooth root. Specifically, we developed a multifunctional peptide-conjugated non-viral gene vector to enhance the expression of vascular endothelial growth factor (VEGF) in hDPSCs, and evaluated the efficiency and bioactivity of the angiogenic transfected hDPSCs in vitro and in a full-length human tooth root model in vivo.
Multifunctional peptide C-R₉-G-NLS-W was conjugated to polylysine (PLL) using click chemistry method. The multifunctional peptide-modified PLL encapsulated pVEGF plasmid using electrostatic interaction and formed polymer/gene complexes. Next, hDPSCs were transfected using the gene complexes. Cellular uptake and transfection efficiency were examined using flow cytometry. PCR was used to evaluate VEGF gene expression of the transfected cells at mRNA level. Furthermore, the effects of transfected DPSCs on the cytotoxicity and endothelial vascular-like tube formation were performed by MTT assay and Matrigel assay in vitro. Transfected DPSCs were injected in human tooth roots sectioned into 11 mm segments and implanted in nude mice. The constructs were retrieved after 4 weeks and examined for regeneration of pulp-like tissue and vascularization by histology.
The MTT result showed that at higher DNA concentration, the cell viability of peptide-modified complexes group was higher than PLL/pVEGF complexes group. The cellular uptake and transfection efficiency of peptide-modified gene complexes was higher than control group. PCR analysis indicated that the mRNA expression after transfected with peptide-modified gene complexes was enhanced. Furthermore, the gene transfection enhanced endothelial cell migration and vascular-like tube formation.
The peptide-conjugated polylysine is an effective non-viral vector for gene delivery and enhanced endothelial cell migration and vascular-like tube formation.
Support: This work was supported by NIH/NIDCR (DE024979)

Due to their biocompatibility and large payload capacity, liposomes are the premier delivery vehicle for hydrophilic therapeutics. However, traditional procedures produce liposomes without size uniformity and stable nanoscale structures, both of which determine efficacy of delivery. We have found that templating the growth of liposomes promotes uniform size and increases the structural stability, as predicted by simulations in Dissipative Particle Dynamics (DPD). We refer to our construct as a Core–Polyethylene-glycol–Lipid Shell nanoparticle (CPLS NP), synthesized through a bottom up approach utilizing an inorganic nanoparticle as a platform from which to scaffold the liposome. Using colloidal Au NPs as the core of the CPLS, a PEG molecule, functionalized with a thiol functional group, self-assembles into a layer on the surface of the Au NP. The PEG molecule is also functionalized with a lipid moiety. Through a second self-assembly, free lipids adsorb on to the templated lipid at the NP surface. The morphology of the lipid shell is determined by the morphology of the core, a phenomenon caused by the template-scaffold. The surface charge can be tuned by incorporating various ionic lipids, a property of traditional liposomes that is retained in the CPLS. The surface charge of the lipid shell can direct cellular uptake of the CPLS NPs. The CPLS NP retains the attractive features of the liposome, an aqueous lumen and direct fusion to a membrane, while also having a predictable volume and morphology with increased stability, important features for drug delivery applications.

Antibody biorecognition forms the basis for numerous biomedical applications such as diagnostic assays, targeted drug delivery and targeted cancer imaging. However, antibodies, especially after being conjugated to surfaces or nanostructures, suffer from stability issues when stored under non-refrigerated conditions. Therefore, enhancing the stability of antibodies on surfaces and nanostructures under ambient and elevated temperatures is of paramount importance for many nanobiotechnology applications. In this study, we introduce a simple and facile approach based on a metal-organic framework (MOF) coating to preserve the biorecognition capability of antibodies immobilized to nanoscale surfaces after exposure to elevated temperatures for a prolonged period. By using atomic force microscopy (AFM)-based force spectroscopy, we demonstrate that the MOF coating is able to preserve the binding force and binding frequency of the anti-CD-146 antibody attached to an AFM tip to CD-146 antigen on the surface of melanoma cells at the single molecule level. We also demonstrate that the MOF coating outperforms another commonly used sucrose coating in terms of maintaining binding force and binding frequency of the antibody to antigen. Herein, the AFM tip functionalized with antibodies provides a nanoscale testbed (analogous to an antibody-conjugated nanostructure) to assess antibody biorecognition at the single molecule level and preservation efficacy under antibody denaturing conditions. This MOF coating approach should be applicable to the preservation of antibody-conjugated nanostructures aiming for targeted drug delivery, targeted cancer imaging, nanobiosensors, and a wide range of applications relying on surface-bound antibodies. The improved stability and elimination of refrigeration requirements will facilitate wide applications of antibody-enabled nanobiotechnology in resource-limited environments and populations.

The transdermal route is attractive for minimal invasive administration of small and large molecular bioactives. Among the transdermal techniques (e.g. iontophoresis, electroporation, jet injectors, ultrasound, ablation, powder injection), micro/nanoneedles are particularly promising because of their straightforward, cost-effective and safe administration. However, precise control the over degradation rate of micro/nanoneedles within the skin remains a great challenge. Here, we introduce porous silicon nanoneedles (pSiNNs) for pain free transdermal drug delivery of biomolecules with controlled degradation rate.
In this work, 40-50 µm pSiNNs were fabricated via deep reactive ion etching with a tip diameter below 1 µm and a diameter of about 8-10 µm. Then, a porous surface was obtained by electrochemical anodisation. It was found that nanoneedle biodegradability and mechanical strength could be tuned by changing the thickness of the porous layer. pSiNNs with a 4.0 µm porous layer can degrade much quicker than 1.6 µm porous layer. In addition, small as well as macromolecular drug molecules could be uniformly loaded into the porous layer of the pSiNN arrays with high drug loading capacity. Ex vivo pig and mouse skin penetration experiment demonstrated that pSiNN can significant increase the transdermal delivery efficacy compared to topical application.
In summary, we have fabricated 40-50 µm pSiNNs with tunable porosity, biodegradability and mechanical strength for transdermal delivery. This platform is able to deliver various biotherapeutics (small molecules to large molecules) through the skin and thereby contributes to innovations in pharmaceutical sciences.

The performances of endocytosis mediated cell uptake of two different types of functional peptides, cell receptor-targeting peptide (CRTP) and cell penetrating peptide (CPP), were analyzed and compared with a common carrier of functional peptides-heavy chain of human ferritin (huHF) nanoparticle. 24 copies of a CPP(TAT peptide from transduction domain of human immunodeficiency virus) and/or a CRTP (functional peptide with specific and strong affinity with either human integrin(αvβ3) or epidermal growth factor receptor I(EGFR)) were genetically presented and exposed on the surface of each huHF nanopariticle. We confirmed both in vitro and in vivo tumor-targeting and endocytosis performance of CRTP-presenting huHF nanoparticles and systematically analyzed the cell targeting, endocytosis, and intracellular localization of recombinant huHF nanoparticles labeled with the in the fluorescent dye (Cy5.5) in vitro cultures of cancer and normal cells. In particular, it is notable that CRTPs led to a uniformly diffused localization of the huHF nanoparticles in cancer cell cytosol, while CPP-mediated endocytosis made the huHF nanoparticles remain in a narrow confined cell endosomal region. These novel and remarkable findings provide highly useful informations to many researchers both in industry and in academia are interested in developing delivery systems/carriers of anticancer drugs.

Carbon Quantum Dots (CQDs) has demonstrated high potency to mitigate neuronal oxidative stress and related pathologies, including Alzheimer's disease (AD). However, the application of CQDs is limited due to their inability to cross the Blood Brain Barrier (BBB) without disrupting the structural integrity of tight junctions. Here, we introduce surface modified CQD nanoencased in ß-lactoglobulin for easy passage across the BBB and displaying both prophylactic and therapeutic efficacy. The diverse ligand binding ability of ß-lactoglobulin was leveraged to encapsulate and transport the surface functionalized CQDs. ß-lactoglobulin owing to its biocompatibility was chosen to improve bioavailability of the encapsulated core. Due to the evidences of the role played by Amyloid-ß to initiate AD, the study employs Amyloid-ß as the model causative species and SH-SY5Y neuroblastoma cell line as the model cell line. Our study illustrates neuroprotective efficacy of the surface functionalized CQDs.

Light triggered switchable nanosystems have recently gained increasing attention as a promising approach of remotely controlled drug delivery. We used the strategy of remotely controlled delivery technology that may overcome the pathophysiological barriers existing in targeted drug delivery. Unlike pathophysiology actived drug targeting strategies, light triggered switchable nanosystems are minimally influenced by the heterogeneity of cells, tissue types, and/or microenvironments. Instead, they are triggered by light (i.e., near-infrared) stimuli, which are absorbed by photoresponsive molecules or nanotransducer. The sequential conversion of light to heat or reactive oxygen species can activate the property change of nanoparticles in a spatio-temporally controlled manner. This talk will present several recent studies of light triggered switchable systems to overcome the pathophysiology barriers and future directions in the application of light-switchable systems for remotely targeted drug delivery.

Service textiles are high-touch surfaces in the healthcare environment and they play an important role in the acquisition and transmission of pathogenic microorganisms, including multi-drug resistant organisms that can survive for weeks on fabrics. Contaminated service textiles have caused infections, which not only affects the safety and health of healthcare personnel and/or patient, but also is incompatible with good hygiene and cross-infection control practices and inconsistent with the expectations of modern healthcare. Here, we use a versatile dip-pad-cure approach to graft/coat polymethacrylamide (PMAA) onto the surfaces of representative service textiles including polyester, cotton, and polyester/cotton blend. Upon treatment with commercial bleach, the amide groups on PMAA were transformed into stable acyclic N-halamines, providing the resultant fabrics with potent, durable (against wash/wear), and rechargeable biocidal activities against E. coli (gram-negative bacteria), S. aureus (gram-positive bacteria), and C. albicans (fungi). The antimicrobial potency of the fabrics could be readily controlled by adjusting the treatment conditions, and the N-halamine contents could be easily determined via a simple chlorine strip test. Further, the comfort performances and the mechanical properties of the antimicrobial fabrics were not adversely affected by the grafting and chlorination treatments. These functions make the N-halamine based antimicrobial fabrics as attractive candidates of service textiles for various healthcare applications.

Herein, we will present the first charge transformable theranostic boron-based nanoparticles for Boron Neutron Capture Therapy (BNCT) and optical imaging. BNCT is an emerging targeted cancer therapy modality that can deliver an immense dose gradient within cellular spatial specificity between tumor and healthy cells. Individually, boron-10 isotope and epithermal neutron flux is not harmful to the body. When combined, boron-10 undergoes a nuclear fission reaction, producing two highly energetic particles in MeV. The alpha-4 and lithium-7 particles deposit all its energy within 5-9 µm in diameter, which corresponds to the size of a single cell. These particles, produced during BNCT treatment, have characteristic of high linear energy transfer (LET) radiation, which induced complex DNA double-strand breaks (DSB). BNCT has showed promising results in clinical trials of patient with recurrent head and neck cancers and enjoyed a good quality of life after receiving two sessions of BNCT treatment. To advance BNCT as one of the cancer treatment modalities, a new and more specific boron drug is needed with superior efficacy compared to currently available boron drug. Specifically, our group is developing the preparation of boron carbon oxynitride (BCNO) nanostructures as a novel theranostic agent, which relies on the inherent luminescence properties of BCNO and the high loading of ¹⁰B within a single nanoparticle for maximizing the efficacy of BNCT treatment. A successful BNCT treatment relies on the delivering a high payload of boron-10 isotopes with high specificity. According to a recent review article on nanoparticle drug delivery, less than 1% of the injected dose of nanoparticle drug reached to the tumor site in small animal studies. This is due to our biological complexity, which demands nanoparticles of different sizes and charges during drug delivery. For examples, for tumor accumulation via EPR effect, the optimal size of the nanoparticles should be between 30-150 nm and possessed a neutral surface charge. Upon accumulation within the tumor microenvironment, a nanoparticle with positive surface charge is known to be effective for cell internalization. Herein, we aim to tackle the latter problem related to the particle surface charge. Positively charge nanoparticles and macromolecules is known to be toxic to cells by forming holes on the cell membrane during internalization and thus resulted in diffusion of cytosolic proteins out of the cells. While this effect is desirable at the cancer cells, the toxic nature of the nanoparticles needs to be first protected with a stealthy polymer ligand until the nanoparticles is delivered to the cancer site. As the first proof of concept, we have successfully synthesized and functionalized BNCO nanostructures with a positively charge polymer followed by a stealthy layer of polyethylene glycol for enhanced tumor accumulation. At the tumor microenvironment, the chemically engineered polymer coating with an acid sensitive bond will break, thus releasing the toxic, and positive charge nanoparticles for effective cell penetration. Previously, our group has successfully showed the functionalization of 5 nm BCNO nanoparticles with polyethyleneimine (PEI) and confirmed the functionality via FTIR and zeta potential measurement. The bare BCNO nanoparticles in water possessed negative charge while the BCNO@PEI possessed a positive charge in water. Herein, we will present the preparation of the block copolymers with an acid reactive linker, nanoparticle functionalization and finally shows the particle charge transformation. In vitro cell toxicity and cell membrane penetration of a series of polymer-coated BCNO nanostructures will also be presented at this meeting.

The fluorescent hybrid nanomaterial systems with inexpensive, transition metal oxide (TMO), such as cobalt oxide and carbon nanomaterials, has gained impressive attention in cellular imaging and viability studies. Surface functionalizing the TMOs with the carbon nanodots (CNDs) leads to synergistic, tunable photoelectronic properties in a hybrid nanoparticle for increasing their photoluminescence intensity, thereby making it a potential bioimaging agent for the cancer cells. By incorporating the cobalt oxide (Co₃O₄) nanoparticles with CNDs, we hypothesize that the surface defect states are altered in a nitrogen-rich carbon nanodots matrix, thereby increasing the surface as well as photoelectronic properties of the hybrid nanoparticles due to the synergistic interfacial properties between the Co₃O₄ and CNDs. Hence, the aim of this research was to synthesize a spherical hybrid with Co₃O₄ nanoparticles on CNDs, which could serve as an efficient bioimaging agent in the cancer cells. A simple, modified microwave technique was carried out for the synthesis of the hybrid nanoparticles. The characterization studies such as UV-Visible, Photoluminescence, Raman, and X-ray photoelectron spectroscopies, X-ray Diffraction, and Transmission electron microscopic techniques were performed for the hybrid nanoparticles. The absorbance and the photoluminescent intensity were observed to be higher for the hybrid nanoparticles than the CNDs due to the incorporation of Co₃O₄ nanoparticles, which may cause structural as well as tunable electronic properties in the hybrid nanomaterial. Cellular uptake and viability studies were performed using A549 and normal human cell line, EA. Hy926. The hybrid nanoparticles were efficiently uptaken by the cancer cells, and a decrease in the cell viability was observed, compared to the human epithelial cells. Overall, the synthesized hybrid nanoparticles can be used as a potential theranostic agent for cancer therapy.

In chronic and burn wounds, novel low-cost therapeutic devices and agents are still sought after in order to actively stimulate skin regeneration at the structural, vascular, and immune levels. Recently, bovine milk extracts have been described to have regenerative capabilities comparable to phenytoin in skin-wound in vivo models [1], thus drawing interest in exploiting this potential. Chitosan films are useful in skin regeneration due to their highly adhesive and antimicrobial properties [2], though their use as protein delivery agents needs to be further explored. Additionally, when supplied cutaneously, lactic acid has been found to promote angiogenesis, collagen deposition, and growth factor production, resulting in accelerated skin regeneration for in vivo models [3]. Here we report the isolation and loading of bovine milk whey proteins onto chitosan-lactic acid films, as well as the evaluation of their chemical, thermal, and mechanical properties and release profiles. The incorporation of chitosan (CS), lactic acid (LA), and milk whey proteins (MWP) is expected to yield low-cost, scalable cutaneous delivery biomaterials with antimicrobial, angiogenic, and immunomodulating capabilities as a result of their respective individual properties.
Milk whey was isolated from commercial pasteurized bovine milk by pH adjustments, centrifugation, and salting out. The protein precipitates were quantified with a Bradford colorimetric assay and analyzed via SDS-PAGE. This analysis revealed that the isolated proteins did not degrade as a result of the process, thus validating the whey isolation protocol as reliable. Gel permeation and cationic exchange chromatographies were performed to confirm the identities of the protein components in the isolated milk whey. The deacetylation degree of the chitosan samples was determined by nuclear magnetic resonance. Dialyzed, freeze-dried milk whey protein isolate was loaded onto chitosan films prepared from chitosan-lactic acid solutions. The resulting CS-LA-MWP films were analyzed using texturometry, FTIR, TGA, and DSC to evaluate film adhesion strength on human skin, elastic modulus, and elongation to break; the incorporation of all components; and the degradation and glass transition temperatures. Protein release kinetics were determined by UV-vis spectroscopy. Antibacterial activity in vitro and water vapor permeability were also determined for CS-LA-MWP films.
Overall, the protein isolation and film preparation processes reported in this work yielded CS-LA-MWP films that were found to be highly elastic and adhesive, while inhibiting bacterial proliferation and releasing their protein load with an appropriate release profile for skin delivery applications. The regenerative potential of these films will be evaluated shortly in murine skin-wound models.

References:
[1] A.A. Hemmati et al. (2018). Int. J. Surg. 54:133-140.
[2] E.I. Rabea et al. (2003). Biomacromolecules. 4(6):1457-1465.
[3] Porporato et al. (2012) Angiogenesis. 15:581-92.

Acknowledgments:
The authors wish to thank Victor Zaldivar-Machorro, Martin Vargas-Suarez, Gerardo Cedillo-Valverde, and Karla Reyes-Morales for technical support in characterization techniques. The authors also thank Maria Cristina Piña-Barba for access to centrifugation and freeze-drying equipment. G.C.S. acknowledges financial support from CONACyT through a graduate studies scholarship. R.V.G. acknowledges financial support awarded by PAPIIT-UNAM through grant IG100220.

Nanomedicine has led to the development of new materials able to improve the pharmaceutical effect of bioactive components, broadening the options of treatment for several diseases like cancer. Chitosan (Cs) and Alginate (ALG) have been firmly established in recent years as biocompatible, biodegradable, mucoadhesive and low-toxic polymers able to form complexes with bioactive agents, making them promising drug delivery vehicles. Some snake venom toxins such as A₂ phospholipases (PLA₂s), serine proteinases (SVSPs) and metalloproteinases (SVMPs) have been reported to present antitumoral activity in different tumor cell-lines, making them a promising option to be used as cancer pharmaceuticals. We identified the major proteins of a black-tailed rattlesnake (Crotalus molossus molossus) venom through MALDI-QTOF analysis. Additionally, the venom against red blood cells and T-47D breast carcinoma cells was evaluated. Afterwards, the snake's venom was loaded into Cs-ALG nanoparticles through the ionotropic gelation process with tripolyphosphate (TPP), obtaining particles of 504.7 ± 22 nm and a Zeta potential of +41 ± 1.6 mV. The Cs-Venom-ALG complex was able to deliver the venom into the breast carcinoma cells through endocytosis, inhibiting their viability and inducing morphological changes in the T-47D cells. Thus, we suggest the potential use of C. m. molossus venom toxins entrapped within polymer nanoparticles for the future development and research of cancer pharmaceuticals

Posttranslational modification and agglomeration of α -Synuclein (α -Syn), mitochondrial dysfunction, oxidative stress and loss of dopaminergic neurons are hallmark of Parkinson’s disease (PD). The α-synucleinopathy is attributed to phosphorylation and aggregation of α-Syn. A strategy to degrade or reduce phosphorylated protein paves the way to develop PD therapy. Hence, nanoformulations of emerging neuroprotective agents, metformin and FTY720 have been evaluated in in vitro and ex vivo experimental PD models. Nature inspired bio-compatible polydopamine nanocarrier for metformin delivery and chitosan nanocarrier for FTY720 has been employed to enhance delivery, brain targeting and bio-availability of drugs. Nanomaterials have shown effective neuroprotection with controlled drug release to improve absorption and bio-availability of drugs. The neuroprotective potential was arbitrated by downregulation of phospho -serine 129 ( pSer129 ) α -Syn, with reduction in oxidative stress, prevention of apoptosis and anti -inflammatory activities. The neuroprotective mechanism uncovered novel physical interaction of protein phosphatase PP2A- polycomb protein EzH2 and EzH2- pSer129 α-Syn to mediate ubiquitination and degradation of agglomerated pSer129 α-Syn. In summary, this study divulges the enhanced neuroprotective role of Met loaded PDANPs and FTY720 nanocomposites by reversing the neurochemical deficits by confirming an epigenetic mediated nanotherapeutic approach for the PD prevention.