Combination Nanomedicine Strategy for Preventing High-Risk Corneal Transplantation Rejection.

High-risk (HR) corneal transplantation presents a formidable challenge, with over 50% of grafts experiencing rejection despite intensive postoperative care involving frequent topical eyedrop administration up to every 2 h, gradually tapering over 6-12 months, and ongoing maintenance dosing. While clinical evidence underscores the potential benefits of inhibiting postoperative angiogenesis, effective antiangiogenesis therapy remains elusive in this context. Here, we engineered controlled-release nanomedicine formulations comprising immunosuppressants (nanoparticles) and antiangiogenesis drugs (nanowafer) and demonstrated that these formulations can prevent HR corneal transplantation rejection for at least 6 months in a clinically relevant rat model. Unlike untreated corneal grafts, which universally faced rejection within 2 weeks postsurgery, a single subconjunctival injection of the long-acting immunosuppressant nanoparticle alone effectively averted graft rejection for 6 months, achieving a graft survival rate of ∼70%. Notably, the combination of an immunosuppressant nanoparticle and an anti-VEGF nanowafer yielded significantly better efficacy with a graft survival rate of >85%. The significantly enhanced efficacy demonstrated that a combination nanomedicine strategy incorporating immunosuppressants and antiangiogenesis drugs can greatly enhance the ocular drug delivery and benefit the outcome of HR corneal transplantation with increased survival rate, ensuring patient compliance and mitigating dosing frequency and toxicity concerns.

Effective intravitreal gene delivery to retinal pigment epithelium with hyaluronic acid nanospheres.

Inherited retinal degeneration (IRD) can cause a wide range of different forms of vision loss and blindness, and in spite of extensive advancements in gene therapy research, therapeutic approaches for targeting IRDs are still lacking. We have recently developed an approach for the intravitreal co-delivery of hyaluronic-acid nanospheres (HA-NSs) with sulfotyrosine (ST), effectively reaching the outer retina from the vitreal cavity. Here, our goal was to understand whether DNA-filled HA-NSs could generate gene expression in the outer retina. TxRed-labeled HA-NSs were compacted with plasmid DNA carrying a GFP reporter gene and intravitreally injected into the mouse retina. Follow-up at 4 weeks showed widespread gene expression in the outer retina and reduced, albeit present, expression at 8 weeks post-injection. Further analysis revealed this expression to be largely localized to the retinal pigment epithelium (RPE). These data show that intravitreal delivery of HA-NSs is a promising non-viral platform for the delivery of therapeutic genes and can generate pan-tissue, persistent gene expression in the RPE.

Noninvasive Delivery of Self-Regenerating Cerium Oxide Nanoparticles to Modulate Oxidative Stress in the Retina.

Diseases affecting the retina, such as age-related macular degeneration (AMD), diabetic retinopathy, macular edema, and retinal vein occlusions, are currently treated by the intravitreal injection of drug formulations. These disease pathologies are driven by oxidative damage due to chronic high concentrations of reactive oxygen species (ROS) in the retina. Intravitreal injections often induce retinal detachment, intraocular hemorrhage, and endophthalmitis. Furthermore, the severe eye pain associated with these injections lead to patient noncompliance and treatment discontinuation. Hence, there is a critical need for the development of a noninvasive therapy that is effective for a prolonged period for treating retinal diseases. In this study, we developed a noninvasive cerium oxide nanoparticle (CNP) delivery wafer (Cerawafer) for the modulation of ROS in the retina. We fabricated Cerawafer loaded with CNP and determined its SOD-like enzyme-mimetic activity and ability to neutralize ROS generated in vitro. We demonstrated Cerawafer's ability to deliver CNP in a noninvasive fashion to the retina in healthy mouse eyes and the CNP retention in the retina for more than a week. Our studies have demonstrated the in vivo efficacy of the Cerawafer to modulate ROS and associated down-regulation of VEGF expression in the retinas of very-low-density lipoprotein receptor knockout (vldlr-/-) mouse model. The development of a Cerawafer nanotherapeutic will fulfill a hitherto unmet need. Currently, there is no such therapeutic available, and the development of a Cerawafer nanotherapeutic will be a major advancement in the treatment of retinal diseases.

3D Printing of MgAl2O4 Spinel Mesh and Densification Through Pressure-Less Sintering and Hot Isostatic Pressing.

MgAl2O4 spinel mesh with micro-features of 410 and 250 µm unit cell length and rib thickness, respectively, was three-dimensional (3D) printed and sintered followed by Hot Isostatic Pressing (HIPing). A stable colloidal dispersion of spinel in polymer-water solution was prepared and 3D-printed using a 30-gauge needle (∼100 µm inner diameter) on a regenHU 3D-Discovery bioprinter. Samples were characterized for their density and microstructure. Samples with near theoretical density after HIPing was subjected to mechanical property evaluation such as hardness by Vickers indentation and elastic modulus using nanoindentation technique. Microstructure of sintered samples across the ribs have shown graded grain structure with finer grains near the edges (0.7 µm average) with occasional porosity and coarser grains toward the center of the rib (5.2 µm average). HIPing resulted in substantial grain growth and the average grain size was found to be 10.9 µm (with a variation in the grain size of 2.2 µm along the edges and 13.1 µm at the center of the rib) exhibiting close packed and dense microstructure. Finer grains toward the edges may probably be due to the flow behavior during printing process and lower distribution of the powder loading along the edges resulting in low green density. This relatively higher porosity pining the grain growth under the extremely low heating rate employed for the controlled shrinkage to maintain the integrity of the sample. 3D printed samples after HIPing exhibited a density of 3.57 g/cc and hardness of 12.95 GPa, which are at par with the samples processed through conventional ceramic processing techniques. Nanoindentation studies employing maximum load of 45 mN with depth have shown an elastic modulus of 238 ± 15 GPa. MgAl2O4 spinel mesh 3D printed in this study is a potential prospective candidate that can be explored for cranioplasty procedures and other biomedical applications.

Dextran Sulfate Polymer Wafer Promotes Corneal Wound Healing.

Eye injuries due to corneal abrasions, chemical spills, penetrating wounds, and microbial infections cause corneal scarring and opacification that result in impaired vision or blindness. However, presently available eye drop formulations of anti-inflammatory and antibiotic drugs are not effective due to their rapid clearance from the ocular surface or due to drug-related side effects such as cataract formation or increased intraocular pressure. In this article, we presented the development of a dextran sulfate-based polymer wafer (DS-wafer) for the effective modulation of inflammation and fibrosis and demonstrated its efficacy in two corneal injury models: corneal abrasion mouse model and alkali induced ocular burn mouse model. The DS-wafers were fabricated by the electrospinning method. We assessed the efficacy of the DS-wafer by light microscopy, qPCR, confocal fluorescence imaging, and histopathological analysis. These studies demonstrated that the DS-wafer treatment is significantly effective in modulating corneal inflammation and fibrosis and inhibited corneal scarring and opacification compared to the unsulfated dextran-wafer treated and untreated corneas. Furthermore, these studies have demonstrated the efficacy of dextran sulfate as an anti-inflammatory and antifibrotic polymer therapeutic.

Development of Antimicrobial Nitric Oxide-Releasing Fibers.

Nitric oxide (NO) is a highly reactive gas molecule, exhibiting antimicrobial properties. Because of its reactive nature, it is challenging to store and deliver NO efficiently as a therapeutic agent. The objective of this study was to develop NO-releasing polymeric fibers (NO-fibers), as an effective delivery platform for NO. NO-fibers were fabricated with biopolymer solutions of polyvinyl pyrrolidone (PVP) and ethylcellulose (EC), and derivatives of N-diazeniumdiolate (NONOate) as NO donor molecules, using an electrospinning system. We evaluated in vitro NO release kinetics, along with antimicrobial effects and cytotoxicity in microorganisms and human cell culture models. We also studied the long-term stability of NONOates in NO-fibers over 12 months. We demonstrated that the NO-fibers could release NO over 24 h, and showed inhibition of the growth of Pseudomonas aeruginosa (P. aeruginosa) and methicillin-resistant Staphylococcus aureus (MRSA), without causing cytotoxicity in human cells. NO-fibers were able to store NONOates for over 12 months at room temperature. This study presents the development of NO-fibers, and the feasibility of NO-fibers to efficiently store and deliver NO, which can be further developed as a bandage.

Co-Injection of Sulfotyrosine Facilitates Retinal Uptake of Hyaluronic Acid Nanospheres Following Intravitreal Injection.

Gene and drug delivery to the retina is a critical therapeutic goal. While the majority of inherited forms of retinal degeneration affect the outer retina, specifically the photoreceptors and retinal pigment epithelium, effective targeted delivery to this region requires invasive subretinal delivery. Our goal in this work was to evaluate two innovative approaches for increasing both the persistence of delivered nanospheres and their penetration into the outer retina while using the much less invasive intravitreal delivery method. We formulated novel hyaluronic acid nanospheres (HA-NS, 250 nm and 500 nm in diameter) conjugated to fluorescent reporters and delivered them intravitreally to the adult Balb/C mouse retina. They exhibited persistence in the vitreous and along the inner limiting membrane (ILM) for up to 30 days (longest timepoint examined) but little retinal penetration. We thus evaluated the ability of the small molecule, sulfotyrosine, to disrupt the ILM, and found that 3.2 µg/µL sulfotyrosine led to significant improvement in delivery to the outer retina following intravitreal injections without causing retinal inflammation, degeneration, or loss of function. Co-delivery of sulfotyrosine and HA-NS led to robust improvements in penetration of HA-NS into the retina and accumulation along the interface between the photoreceptors and the retinal pigment epithelium. These exciting findings suggest that sulfotyrosine and HA-NS may be an effective strategy for outer retinal targeting after intravitreal injection.

CD8+ T cells inhibit metastasis and CXCL4 regulates its function.

BACKGROUND: The mechanism by which immune cells regulate metastasis is unclear. Understanding the role of immune cells in metastasis will guide the development of treatments improving patient survival. METHODS: We used syngeneic orthotopic mouse tumour models (wild-type, NOD/scid and Nude), employed knockout (CD8 and CD4) models and administered CXCL4. Tumours and lungs were analysed for cancer cells by bioluminescence, and circulating tumour cells were isolated from blood. Immunohistochemistry on the mouse tumours was performed to confirm cell type, and on a tissue microarray with 180 TNBCs for human relevance. TCGA data from over 10,000 patients were analysed as well. RESULTS: We reveal that intratumoral immune infiltration differs between metastatic and non-metastatic tumours. The non-metastatic tumours harbour high levels of CD8+ T cells and low levels of platelets, which is reverse in metastatic tumours. During tumour progression, platelets and CXCL4 induce differentiation of monocytes into myeloid-derived suppressor cells (MDSCs), which inhibit CD8+ T-cell function. TCGA pan-cancer data confirmed that CD8lowPlatelethigh patients have a significantly lower survival probability compared to CD8highPlateletlow. CONCLUSIONS: CD8+ T cells inhibit metastasis. When the balance between CD8+ T cells and platelets is disrupted, platelets produce CXCL4, which induces MDSCs thereby inhibiting the CD8+ T-cell function.

3D-Bioprinted Inflammation Modulating Polymer Scaffolds for Soft Tissue Repair.

Development of inflammation modulating polymer scaffolds for soft tissue repair with minimal postsurgical complications is a compelling clinical need. However, the current standard of care soft tissue repair meshes for hernia repair is highly inflammatory and initiates a dysregulated inflammatory process causing visceral adhesions and postsurgical complications. Herein, the development of an inflammation modulating biomaterial scaffold (bioscaffold) for soft tissue repair is presented. The bioscaffold design is based on the idea that, if the excess proinflammatory cytokines are sequestered from the site of injury by the surgical implantation of a bioscaffold, the inflammatory response can be modulated, and the visceral adhesion formations and postsurgical complications can be minimized. The bioscaffold is fabricated by 3D-bioprinting of an in situ phosphate crosslinked poly(vinyl alcohol) polymer. In vivo efficacy of the bioscaffold is evaluated in a rat ventral hernia model. In vivo proinflammatory cytokine expression analysis and histopathological analysis of the tissues have confirmed that the bioscaffold acts as an inflammation trap and captures the proinflammatory cytokines secreted at the implant site and effectively modulates the local inflammation without the need for exogenous anti-inflammatory agents. The bioscaffold is very effective in inhibiting visceral adhesions formation and minimizing postsurgical complications.

Challenges and opportunities for drug delivery to the posterior of the eye.

Drug delivery to the posterior segment of the eye remains challenging even though the eye is readily accessible. Its unique and complex anatomy and physiology contribute to the limited options for drug delivery via non-invasive topical treatment, which is the prevalent ophthalmic treatment. To treat the most common retinal diseases, intravitreal (IVT) injection has been a common and effective therapy. With the advancement of nanotechnologies, novel formulations and drug delivery systems are being developed to treat posterior segment diseases. Here, we discuss the recent advancement in ocular delivery systems, including-sustained release formulations, IVT implants, and preclinical topical formulations, and the challenges faced in their clinical translation.

Transcutaneously refillable, 3D-printed biopolymeric encapsulation system for the transplantation of endocrine cells.

Autologous cell transplantation holds enormous promise to restore organ and tissue functions in the treatment of various pathologies including endocrine, cardiovascular, and neurological diseases among others. Even though immune rejection is circumvented with autologous transplantation, clinical adoption remains limited due to poor cell retention and survival. Cell transplant success requires homing to vascularized environment, cell engraftment and importantly, maintenance of inherent cell function. To address this need, we developed a three dimensional (3D) printed cell encapsulation device created with polylactic acid (PLA), termed neovascularized implantable cell homing and encapsulation (NICHE). In this paper, we present the development and systematic evaluation of the NICHE in vitro, and the in vivo validation with encapsulated testosterone-secreting Leydig cells in Rag1-/- castrated mice. Enhanced subcutaneous vascularization of NICHE via platelet-rich plasma (PRP) hydrogel coating and filling was demonstrated in vivo via a chorioallantoic membrane (CAM) assay as well as in mice. After establishment of a pre-vascularized bed within the NICHE, transcutaneously transplanted Leydig cells, maintained viability and robust testosterone secretion for the duration of the study. Immunohistochemical analysis revealed extensive Leydig cell colonization in the NICHE. Furthermore, transplanted cells achieved physiologic testosterone levels in castrated mice. The promising results provide a proof of concept for the NICHE as a viable platform technology for autologous cell transplantation for the treatment of a variety of diseases.

Application of Hydrogel Template Strategy in Ocular Drug Delivery.

The hydrogel template strategy was previously developed to fabricate homogeneous polymeric microparticles. Here, we demonstrate the versatility of the hydrogel template strategy for the development of nanowafer-based ocular drug delivery systems. We describe the fabrication of dexamethasone-loaded nanowafers using polyvinyl alcohol and the instillation of a nanowafer on a mouse eye. The nanowafer, a small circular disk, is placed on the ocular surface, and it releases a drug as it slowly dissolves over time, thus increasing ocular bioavailability and enhancing efficiency to treat eye injuries.

Synergistic Cysteamine Delivery Nanowafer as an Efficacious Treatment Modality for Corneal Cystinosis.

A synergy between the polymer biomaterial and drug plays an important role in enhancing the therapeutic efficacy, improving the drug stability, and minimizing the local immune responses in the development of drug delivery systems. Particularly, in the case of ocular drug delivery, the need for the development of synergistic drug delivery system becomes more pronounced because of the wet ocular mucosal surface and highly innervated cornea, which elicit a strong inflammatory response to the instilled drug formulations. This article presents the development of a synergistic cysteamine delivery nanowafer to treat corneal cystinosis. Corneal cystinosis is a rare metabolic disease that causes the accumulation of cystine crystals in the cornea resulting in corneal opacity and loss of vision. It is treated with topical cysteamine (Cys) eye drops that need to be instilled 6-12 times a day throughout the patient's life, which causes side effects such as eye pain, redness, and ocular inflammation. As a result, compliance and treatment outcomes are severely compromised. To surmount these issues, we have developed a clinically translatable Cys nanowafer (Cys-NW) that can be simply applied on the eye with a fingertip. During the course of the drug release, Cys-NW slowly dissolves and fades away. The in vivo studies in cystinosin knockout mice demonstrated twice the therapeutic efficacy of Cys-NW containing 10 µg of Cys administered once a day, compared to 44 µg of Cys as topical eye drops administered twice a day. Furthermore, Cys-NW stabilizes Cys for up to four months at room temperature compared to topical Cys eye drops that need to be frozen or refrigerated and still remain active for only 1 week. The Cys-NW, because of its enhanced therapeutic efficacy, safety profile, and extended drug stability at room temperature, can be rapidly translated to the clinic for human trials.

Dexamethasone Drug Eluting Nanowafers Control Inflammation in Alkali-Burned Corneas Associated With Dry Eye.

PURPOSE: To evaluate the efficacy of a controlled release dexamethasone delivery system for suppressing inflammation in an ocular burn + desiccating stress (OB+DS) model. METHODS: Nanowafers (NW) loaded with Dexamethasone (Dex, 10 µg) or vehicles (2.5% Methylcellulose; MC) were fabricated using hydrogel template strategy. C57BL/6 mice were subjected to unilateral alkali ocular burn with concomitant desiccating stress for 2 or 5 days and topically treated either with 2 µL of 0.1% Dex or vehicle four times per day and compared with mice that had MC-NW or Dex-NW placed on their corneas. Clinical parameters were evaluated daily. Mice were euthanized after 2 or 5 days. Quantitative PCR evaluated the expression of inflammatory cytokines IL-1ß and IL-6 and matrix metalloproteinases (MMP) in whole cornea lysates. Myeloperoxidase activity (MPO) was measured using a commercial kit in cornea lysates. RESULTS: Both Dex drop and Dex-NW groups had significantly lower corneal opacity scores compared with their vehicles. Both Dex drops and Dex-NW significantly decreased expression of IL-1ß, IL-6, and MMP-9 RNA transcripts compared with vehicle drops or wafers 2 and 5 days after the initial lesion. A significant lower number of neutrophils was found in both Dex treatment groups and this was accompanied by decreased MPO activity compared with vehicle controls. CONCLUSIONS: Dex-NW has efficacy equal to Dex drops in preserving corneal clarity and decreasing expression of MMPs and inflammatory cytokines of the corneas of mice subjected to an OB+DS model.

Porcine acellular lung matrix for wound healing and abdominal wall reconstruction: A pilot study.

Surgical wound healing applications require bioprosthetics that promote cellular infiltration and vessel formation, metrics associated with increased mechanical strength and resistance to infection. Porcine acellular lung matrix is a novel tissue scaffold known to promote cell adherence while minimizing inflammatory reactions. In this study, we evaluate the capacity of porcine acellular lung matrix to sustain cellularization and neovascularization in a rat model of subcutaneous implantation and chronic hernia repair. We hypothesize that, compared to human acellular dermal matrix, porcine acellular lung matrix would promote greater cell infiltration and vessel formation. Following pneumonectomy, porcine lungs were processed and characterized histologically and by scanning electron microscopy to demonstrate efficacy of the decellularization. Using a rat model of subcutaneou implantation, porcine acellular lung matrices (n = 8) and human acellular dermal matrices (n = 8) were incubated in vivo for 6 weeks. To evaluate performance under mechanically stressed conditions, porcine acellular lung matrices (n = 7) and human acellular dermal matrices (n = 7) were implanted in a rat model of chronic ventral incisional hernia repair for 6 weeks. After 6 weeks, tissues were evaluated using hematoxylin and eosin and Masson's trichrome staining to quantify cell infiltration and vessel formation. Porcine acellular lung matrices were shown to be successfully decellularized. Following subcutaneous implantation, macroscopic vessel formation was evident. Porcine acellular lung matrices demonstrated sufficient incorporation and showed no evidence of mechanical failure after ventral hernia repair. Porcine acellular lung matrices demonstrated significantly greater cellular density and vessel formation when compared to human acellular dermal matrix. Vessel sizes were similar across all groups. Cell infiltration and vessel formation are well-characterized metrics of incorporation associated with improved surgical outcomes. Porcine acellular lung matrices are a novel class of acellular tissue scaffold. The increased cell and vessel density may promote long-term improved incorporation and mechanical properties. These findings may be due to the native lung scaffold architecture guiding cell migration and vessel formation. Porcine acellular lung matrices represent a new alternative for surgical wound healing applications where increased cell density and vessel formation are sought.

Dexamethasone nanowafer as an effective therapy for dry eye disease.

Dry eye disease is a major public health problem that affects millions of people worldwide. It is presently treated with artificial tear and anti-inflammatory eye drops that are generally administered several times a day and may have limited therapeutic efficacy. To improve convenience and efficacy, a dexamethasone (Dex) loaded nanowafer (Dex-NW) has been developed that can release the drug on the ocular surface for a longer duration of time than drops, during which it slowly dissolves. The Dex-NW was fabricated using carboxymethyl cellulose polymer and contains arrays of 500 nm square drug reservoirs filled with Dex. The in vivo efficacy of the Dex-NW was evaluated using an experimental mouse dry eye model. These studies demonstrated that once a day Dex-NW treatment on alternate days during a five-day treatment period was able to restore a healthy ocular surface and corneal barrier function with comparable efficacy to twice a day topically applied dexamethasone eye drop treatment. The Dex-NW was also very effective in down regulating expression of inflammatory cytokines (TNF-α, and IFN-γ), chemokines (CXCL-10 and CCL-5), and MMP-3, that are stimulated by dry eye. Despite less frequent dosing, the Dex-NW has comparable therapeutic efficacy to topically applied Dex eye drops in experimental mouse dry eye model, and these results provide a strong rationale for translation to human clinical trials for dry eye.

Ocular drug delivery nanowafer with enhanced therapeutic efficacy.

Presently, eye injuries are treated by topical eye drop therapy. Because of the ocular surface barriers, topical eye drops must be applied several times in a day, causing side effects such as glaucoma, cataract, and poor patient compliance. This article presents the development of a nanowafer drug delivery system in which the polymer and the drug work synergistically to elicit an enhanced therapeutic efficacy with negligible adverse immune responses. The nanowafer is a small transparent circular disc that contains arrays of drug-loaded nanoreservoirs. The slow drug release from the nanowafer increases the drug residence time on the ocular surface and its subsequent absorption into the surrounding ocular tissue. At the end of the stipulated period of drug release, the nanowafer will dissolve and fade away. The in vivo efficacy of the axitinib-loaded nanowafer was demonstrated in treating corneal neovascularization (CNV) in a murine ocular burn model. The laser scanning confocal imaging and RT-PCR study revealed that once a day administered axitinib nanowafer was therapeutically twice as effective, compared to axitinib delivered twice a day by topical eye drop therapy. The axitinib nanowafer is nontoxic and did not affect the wound healing and epithelial recovery of the ocular burn induced corneas. These results confirmed that drug release from the axitinib nanowafer is more effective in inhibiting CNV compared to the topical eye drop treatment even at a lower dosing frequency.

Hydrogel templates for the fabrication of homogeneous polymer microparticles.

Nano/microparticulate drug delivery systems with homogeneous size distribution and predefined shape are important in understanding the influence of the geometry and dimensions of these systems on blood circulation times and cellular uptake. We present a general method using water dissolvable hydrogel templates for the fabrication of homogeneous, shape-specific polymer/drug constructs in the size range of 200 nm to 50 µm. This hydrogel template strategy is mild, inexpensive, and readily scalable for the fabrication of multifunctional drug delivery vehicles.

A study of drug release from homogeneous PLGA microstructures.

The hydrogel template method was used to fabricate homogeneous drug-PLGA microparticles. Four drugs (felodipine, risperidone, progesterone, and paclitaxel) were loaded into the PLGA particles with the homogeneous size of 10microm, 20microm, and 50microm. The drug loading into the PLGA microparticles was 50% and higher. The felodipine-PLGA microstructures of four different sizes showed that the drug release kinetics is dependent on the total surface area available for drug release. The smaller the particle size, the release rate was faster. Two types of microparticles (10microm diameter and 10microm height, and 50microm diameter and 5microm height) showed zero-order release and complete release was observed within 2weeks. The release rate, however, was not exactly proportional to the surface area. Different drugs which were loaded into the same PLGA formulation showed different release profiles. The main difference was on the initial burst release. The overall release profile seems to be similar for different drugs, if the release profile is adjusted to eliminate the burst release. The initial burst release appears to be inversely related to the water-solubility of a drug, i.e., the lower the water-solubility of a drug, the higher the burst release. The hydrogel template method allowed preparation of homogeneous particles with predefined sizes with high drug loading. It allowed study on the effect of size and shape on the drug release kinetics. With the microparticles of homogeneous size and shape, the drug release kinetics can be projected based on the size of microparticles and water-solubility of a drug. The ability of making homogeneous particles is expected to provide better prediction and reproducibility of the drug release property of a given formulation.

The hydrogel template method for fabrication of homogeneous nano/microparticles.

Nano/microparticles have been used widely in drug delivery applications. The majority of the particles are prepared by the conventional emulsion methods, which tend to result in particles with heterogeneous size distribution with sub-optimal drug loading and release properties. Recently, microfabrication methods have been used to make nano/microparticles with a monodisperse size distribution. The existing methods utilize solid templates for making particles, and the collection of individual particles after preparation has not been easy. The new hydrogel template approach was developed to make the particle preparation process simple and fast. The hydrogel template approach is based on the unique properties of physical gels that can undergo sol-gel phase transition upon changes in environmental conditions. The phase reversible hydrogels, however, are in general mechanically too weak to be treated as a solid material. It was unexpectedly found that gelatin hydrogels could be made to possess various properties necessary for microfabrication of nano/microparticles in large quantities. The size of the particles can be adjusted from 200 nm to >50 microm, providing flexibility in controlling the size in drug delivery formulations. The simplicity in processing makes the hydrogel template method useful for scale-up manufacturing of particles. The drug loading capacity is 50% or higher, and yet the initial burst release is minimal. The hydrogel template approach presents a new strategy of preparing nano/microparticles of predefined size and shape with homogeneous size distribution for drug delivery applications.