Roll-to-roll, high-resolution 3D printing of shape-specific particles.

Particle fabrication has attracted recent attention owing to its diverse applications in bioengineering1,2, drug and vaccine delivery3-5, microfluidics6,7, granular systems8,9, self-assembly5,10,11, microelectronics12,13 and abrasives14. Herein we introduce a scalable, high-resolution, 3D printing technique for the fabrication of shape-specific particles based on roll-to-roll continuous liquid interface production (r2rCLIP). We demonstrate r2rCLIP using single-digit, micron-resolution optics in combination with a continuous roll of film (in lieu of a static platform), enabling the rapidly permutable fabrication and harvesting of shape-specific particles from a variety of materials and with complex geometries, including geometries not possible to achieve with advanced mould-based techniques. We demonstrate r2rCLIP production of mouldable and non-mouldable shapes with voxel sizes as small as 2.0 × 2.0 µm2 in the print plane and 1.1 ± 0.3 µm unsupported thickness, at speeds of up to 1,000,000 particles per day. Such microscopic particles with permutable, intricate designs enable direct integration within biomedical, analytical and advanced materials applications.

Growing three-dimensional objects with light.

Vat photopolymerization (VP) additive manufacturing enables fabrication of complex 3D objects by using light to selectively cure a liquid resin. Developed in the 1980s, this technique initially had few practical applications due to limitations in print speed and final part material properties. In the four decades since the inception of VP, the field has matured substantially due to simultaneous advances in light delivery, interface design, and materials chemistry. Today, VP materials are used in a variety of practical applications and are produced at industrial scale. In this perspective, we trace the developments that enabled this printing revolution by focusing on the enabling themes of light, interfaces, and materials. We focus on these fundamentals as they relate to continuous liquid interface production (CLIP), but provide context for the broader VP field. We identify the fundamental physics of the printing process and the key breakthroughs that have enabled faster and higher-resolution printing, as well as production of better materials. We show examples of how in situ print process monitoring methods such as optical coherence tomography can drastically improve our understanding of the print process. Finally, we highlight areas of recent development such as multimaterial printing and inorganic material printing that represent the next frontiers in VP methods.

Transdermal vaccination via 3D-printed microneedles induces potent humoral and cellular immunity.

Vaccination is an essential public health measure for infectious disease prevention. The exposure of the immune system to vaccine formulations with the appropriate kinetics is critical for inducing protective immunity. In this work, faceted microneedle arrays were designed and fabricated utilizing a three-dimensional (3D)-printing technique called continuous liquid interface production (CLIP). The faceted microneedle design resulted in increased surface area as compared with the smooth square pyramidal design, ultimately leading to enhanced surface coating of model vaccine components (ovalbumin and CpG). Utilizing fluorescent tags and live-animal imaging, we evaluated in vivo cargo retention and bioavailability in mice as a function of route of delivery. Compared with subcutaneous bolus injection of the soluble components, microneedle transdermal delivery not only resulted in enhanced cargo retention in the skin but also improved immune cell activation in the draining lymph nodes. Furthermore, the microneedle vaccine induced a potent humoral immune response, with higher total IgG (Immunoglobulin G) and a more balanced IgG1/IgG2a repertoire and achieved dose sparing. Furthermore, it elicited T cell responses as characterized by functional cytotoxic CD8+ T cells and CD4+ T cells secreting Th1 (T helper type 1)-cytokines. Taken together, CLIP 3D-printed microneedles coated with vaccine components provide a useful platform for a noninvasive, self-applicable vaccination.

Difference between approximate and rigorously measured transference numbers in fluorinated electrolytes.

The performance of binary electrolytes is governed by three transport properties: conductivity, salt diffusion coefficient, and transference number. Rigorous methods for measuring conductivity and the salt diffusion coefficient are well established and used routinely in the literature. The commonly used methods for measuring transference number are the steady-state current method, t+,id, and pulsed field gradient NMR, t+,NMR. These methods yield the transference number only if the electrolyte is ideal, i.e., the salt dissociates completely into non-interacting anions and cations. In this work, we present a complete set of ion transport properties for mixtures of a functionalized perfluoroether, dimethyl carbonate terminated perfluorinated tetraethylene ether, and lithium bis(fluorosulfonyl)imide (LiFSI). The equations used to determine these properties from experimental data are based on Newman's concentrated solution theory. The concentrated-solution-theory-based transference number, t, is negative across all salt concentrations, and it increases with increasing salt concentration. In contrast, the ideal transference number, t+,id, is positive across all salt concentrations and it decreases with salt concentration. The NMR-based transference number, t+,NMR, is approximately 0.5, independent of salt concentration. The disparity between the three transference numbers, which indicates the dominance of ion clustering, is resolved by the use of Newman's concentrated solution theory.

Layerless fabrication with continuous liquid interface production.

Despite the increasing popularity of 3D printing, also known as additive manufacturing (AM), the technique has not developed beyond the realm of rapid prototyping. This confinement of the field can be attributed to the inherent flaws of layer-by-layer printing and, in particular, anisotropic mechanical properties that depend on print direction, visible by the staircasing surface finish effect. Continuous liquid interface production (CLIP) is an alternative approach to AM that capitalizes on the fundamental principle of oxygen-inhibited photopolymerization to generate a continual liquid interface of uncured resin between the growing part and the exposure window. This interface eliminates the necessity of an iterative layer-by-layer process, allowing for continuous production. Herein we report the advantages of continuous production, specifically the fabrication of layerless parts. These advantages enable the fabrication of large overhangs without the use of supports, reduction of the staircasing effect without compromising fabrication time, and isotropic mechanical properties. Combined, these advantages result in multiple indicators of layerless and monolithic fabrication using CLIP technology.

Compliant glass-polymer hybrid single ion-conducting electrolytes for lithium batteries.

Despite high ionic conductivities, current inorganic solid electrolytes cannot be used in lithium batteries because of a lack of compliance and adhesion to active particles in battery electrodes as they are discharged and charged. We have successfully developed a compliant, nonflammable, hybrid single ion-conducting electrolyte comprising inorganic sulfide glass particles covalently bonded to a perfluoropolyether polymer. The hybrid with 23 wt% perfluoropolyether exhibits low shear modulus relative to neat glass electrolytes, ionic conductivity of 10(-4) S/cm at room temperature, a cation transference number close to unity, and an electrochemical stability window up to 5 V relative to Li(+)/Li. X-ray absorption spectroscopy indicates that the hybrid electrolyte limits lithium polysulfide dissolution and is, thus, ideally suited for Li-S cells. Our work opens a previously unidentified route for developing compliant solid electrolytes that will address the challenges of lithium batteries.

Iontophoretic device delivery for the localized treatment of pancreatic ductal adenocarcinoma.

Poor delivery and systemic toxicity of many cytotoxic agents, such as the recent promising combination chemotherapy regimen of folinic acid (leucovorin), fluorouracil, irinotecan, and oxaliplatin (FOLFIRINOX), restrict their full utility in the treatment of pancreatic cancer. Local delivery of chemotherapies has become possible using iontophoretic devices that are implanted directly onto pancreatic tumors. We have fabricated implantable iontophoretic devices and tested the local iontophoretic delivery of FOLFIRINOX for the treatment of pancreatic cancer in an orthotopic patient-derived xenograft model. Iontophoretic delivery of FOLFIRINOX was found to increase tumor exposure by almost an order of magnitude compared with i.v. delivery with substantially lower plasma concentrations. Mice treated for 7 wk with device FOLFIRINOX experienced significantly greater tumor growth inhibition compared with i.v. FOLFIRINOX. A marker of cell proliferation, Ki-67, was stained, showing a significant reduction in tumor cell proliferation. These data capitalize on the unique ability of an implantable iontophoretic device to deliver much higher concentrations of drug to the tumor compared with i.v. delivery. Local iontophoretic delivery of cytotoxic agents should be considered for the treatment of patients with unresectable nonmetastatic disease and for patients with the need for palliation of local symptoms, and may be considered as a neoadjuvant approach to improve resection rates and outcome in patients with localized and locally advanced pancreatic cancer.

Formulation of High-Performance Dry Powder Aerosols for Pulmonary Protein Delivery.

PURPOSE: Pulmonary delivery of biologics is of great interest, as it can be used for the local treatment of respiratory diseases or as a route to systemic drug delivery. To reach the full potential of inhaled biologics, a formulation platform capable of producing high performance aerosols without altering protein native structure is required. METHODS: A formulation strategy using Particle Replication in Non-wetting Templates (PRINT) was developed to produce protein dry powders with precisely engineered particle morphology. Stability of the incorporated proteins was characterized and the aerosol properties of the protein dry powders was evaluated in vitro with an Andersen Cascade Impactor (ACI). RESULTS: Model proteins bovine serum albumin (BSA) and lysozyme were micromolded into 1 µm cylinders composed of more than 80% protein, by mass. Extensive characterization of the incorporated proteins found no evidence of alteration of native structures. The BSA formulation produced a mass median aerodynamic diameter (MMAD) of 1.77 µm ± 0.06 and a geometric standard deviation (GSD) of 1.51 ± 0.06 while the lysozyme formulation had an MMAD of 1.83 µm ± 0.12 and a GSD of 1.44 ± 0.03. CONCLUSION: Protein dry powders manufactured with PRINT could enable high-performance delivery of protein therapeutics to the lungs.

Controlled analysis of nanoparticle charge on mucosal and systemic antibody responses following pulmonary immunization.

Pulmonary immunization enhances local humoral and cell-mediated mucosal protection, which are critical for vaccination against lung-specific pathogens such as influenza or tuberculosis. A variety of nanoparticle (NP) formulations have been tested preclinically for pulmonary vaccine development, yet the role of NP surface charge on downstream immune responses remains poorly understood. We used the Particle Replication in Non-Wetting Templates (PRINT) process to synthesize hydrogel NPs that varied only in surface charge and otherwise maintained constant size, shape, and antigen loading. Pulmonary immunization with ovalbumin (OVA)-conjugated cationic NPs led to enhanced systemic and lung antibody titers compared with anionic NPs. Increased antibody production correlated with robust germinal center B-cell expansion and increased activated CD4(+) T-cell populations in lung draining lymph nodes. Ex vivo treatment of dendritic cells (DCs) with OVA-conjugated cationic NPs induced robust antigen-specific T-cell proliferation with ∼ 100-fold more potency than soluble OVA alone. Enhanced T-cell expansion correlated with increased expression of surface MHCII, T-cell coactivating receptors, and key cytokines/chemokine expression by DCs treated with cationic NPs, which were not observed with anionic NPs or soluble OVA. Together, these studies highlight the importance of NP surface charge when designing pulmonary vaccines, and our findings support the notion that cationic NP platforms engender potent humoral and mucosal immune responses.

Mediating Passive Tumor Accumulation through Particle Size, Tumor Type, and Location.

As the enhanced permeation and retention (EPR) effect continues to be a controversial topic in nanomedicine, we sought to examine EPR as a function of nanoparticle size, tumor model, and tumor location, while also evaluating tumors for EPR mediating factors such as microvessel density, vascular permeability, lymphatics, stromal content, and tumor-associated immune cells. Tumor accumulation was evaluated for 55 × 60, 80 × 180, and 80 × 320 nm PRINT particles in four subcutaneous flank tumor models (SKOV3 human ovarian, 344SQ murine nonsmall cell lung, A549 human nonsmall cell lung, and A431 human epidermoid cancer). Each tumor model revealed specific particle accumulation trends with evident particle size dependence. Immuno-histochemistry staining revealed differences in tumor microvessel densities that correlated with overall tumor accumulation. Immunofluorescence images displayed size-mediated tumor penetration with signal from the larger particles concentrated close to the blood vessels, while signal from the smaller particle was observed throughout the tissue. Differences were also observed for the 55 × 60 nm particle tumor penetration across flank tumor models as a function of stromal content. The 55 × 60 nm particles were further evaluated in three orthotopic, metastatic tumor models (344SQ, A549, and SKOV3), revealing preferential accumulation in primary tumors and metastases over healthy tissue. Moreover, we observed higher tumor accumulation in the orthotopic lung cancer models than in the flank lung cancer models, whereas tumor accumulation was constant for both orthotopic and flank ovarian cancer models, further demonstrating the variability in the EPR effect as a function of tumor model and location.

Docetaxel-Loaded PLGA Nanoparticles Improve Efficacy in Taxane-Resistant Triple-Negative Breast Cancer.

Novel treatment strategies, including nanomedicine, are needed for improving management of triple-negative breast cancer. Patients with triple-negative breast cancer, when considered as a group, have a worse outcome after chemotherapy than patients with breast cancers of other subtypes, a finding that reflects the intrinsically adverse prognosis associated with the disease. The aim of this study was to improve the efficacy of docetaxel by incorporation into a novel nanoparticle platform for the treatment of taxane-resistant triple-negative breast cancer. Rod-shaped nanoparticles encapsulating docetaxel were fabricated using an imprint lithography based technique referred to as Particle Replication in Nonwetting Templates (PRINT). These rod-shaped PLGA-docetaxel nanoparticles were tested in the C3(1)-T-antigen (C3Tag) genetically engineered mouse model (GEMM) of breast cancer that represents the basal-like subtype of triple-negative breast cancer and is resistant to therapeutics from the taxane family. This GEMM recapitulates the genetics of the human disease and is reflective of patient outcome and, therefore, better represents the clinical impact of new therapeutics. Pharmacokinetic analysis showed that delivery of these PLGA-docetaxel nanoparticles increased docetaxel circulation time and provided similar docetaxel exposure to tumor compared to the clinical formulation of docetaxel, Taxotere. These PLGA-docetaxel nanoparticles improved tumor growth inhibition and significantly increased median survival time. This study demonstrates the potential of nanotechnology to improve the therapeutic index of chemotherapies and rescue therapeutic efficacy to treat nonresponsive cancers.

Mechanism of ion transport in perfluoropolyether electrolytes with a lithium salt.

Perfluoropolyethers (PFPEs) are polymer electrolytes with fluorinated carbon backbones that have high flash points and have been shown to exhibit moderate conductivities and high cation transference numbers when mixed with lithium salts. Ion transport in four PFPE electrolytes with different endgroups was characterized by differential scanning calorimetry (DSC), ac impedance, and pulsed-field gradient NMR (PFG-NMR) as a function of salt concentration and temperature. In spite of the chemical similarity of the electrolytes, salt diffusion coefficients measured by PFG-NMR and the glass transition temperature measured by DSC appear to be uncorrelated to ionic conductivity measured by ac impedance. We calculate a non-dimensional parameter, ß, that depends on the salt diffusion coefficients and ionic conductivity. We also use the Vogel-Tammann-Fulcher relationship to fit the temperature dependence of conductivity. We present a linear relationship between the prefactor in the VTF fit and ß; both parameters vary by four orders of magnitude in our experimental window. Our analysis suggests that transport in electrolytes with low dielectric constants (low ß) is dictated by ion hopping between clusters.

Incipient microphase separation in short chain perfluoropolyether-block-poly(ethylene oxide) copolymers.

Incipient microphase separation is observed by wide angle X-ray scattering (WAXS) in short chain multiblock copolymers consisting of perfluoropolyether (PFPE) and poly(ethylene oxide) (PEO) segments. Two PFPE-PEO block copolymers were studied; one with dihydroxyl end groups and one with dimethyl carbonate end groups. Despite having a low degree of polymerization (N ∼ 10), these materials exhibited significant scattering intensity, due to disordered concentration fluctuations between their PFPE-rich and PEO-rich domains. The disordered scattering intensity was fit to a model based on a multicomponent random phase approximation to determine the value of the interaction parameter, χ, and the radius of gyration, Rg. Over the temperature range 30-90 °C, the values of χ were determined to be very large (∼2-2.5), indicating a high degree of immiscibility between the PFPE and PEO blocks. In PFPE-PEO, due to the large electron density contrast between the fluorinated and non-fluorinated block and the high value of χ, disordered scattering was detected at intermediate scattering angles, (q ∼ 2 nm-1) for relatively small polymer chains. Our ability to detect concentration fluctuations was enabled by both a relatively large value of χ and significant scattering contrast.

Nonflammable perfluoropolyether-based electrolytes for lithium batteries.

The flammability of conventional alkyl carbonate electrolytes hinders the integration of large-scale lithium-ion batteries in transportation and grid storage applications. In this study, we have prepared a unique nonflammable electrolyte composed of low molecular weight perfluoropolyethers and bis(trifluoromethane)sulfonimide lithium salt. These electrolytes exhibit thermal stability beyond 200 °C and a remarkably high transference number of at least 0.91 (more than double that of conventional electrolytes). Li/LiNi1/3Co1/3Mn1/3O2 cells made with this electrolyte show good performance in galvanostatic cycling, confirming their potential as rechargeable lithium batteries with enhanced safety and longevity.

Reduction Sensitive PEG Hydrogels for Codelivery of Antigen and Adjuvant To Induce Potent CTLs.

Educating our immune system via vaccination is an attractive approach to combat infectious diseases. Eliciting antigen specific cytotoxic T cells (CTLs), CD8+ effector T cells, is essential in controlling intracellular infectious diseases such as influenza (Flu), tuberculosis (TB), hepatitis, and HIV/AIDS, as well as tumors. However, vaccination utilizing subunit peptides to elicit a potent CD8+ T cell response with antigenic peptides is typically ineffective due to poor immunogenicity. Here we have engineered a reduction sensitive nanoparticle (NP) based subunit vaccine for intracellular delivery of an antigenic peptide and immunostimulatory adjuvant. We have co-conjugated an antigenic peptide (ovalbumin-derived CTL epitope [OVA257-264: SIINFEKL]) and an immunostimulatory adjuvant (CpG ODNs, TLR9 agonist) to PEG hydrogel NPs via a reduction sensitive linker. Bone-marrow derived dendritic cells (BMDCs) treated with the SIINFEKL conjugated NPs efficiently cross-presented the antigenic peptide via MHC-I surface receptor and induced proliferation of OT-I T cells. CpG ODN-conjugated NPs induced maturation of BMDCs as evidenced by the overexpression of CD80 and CD40 costimulatory receptors. Moreover, codelivery of NP conjugated SIINFEKL and CpG ODN significantly increased the frequency of IFN-γ producing CD8+ effector T cells in mice (∼6-fold improvement over soluble antigen and adjuvant). Furthermore, the NP subunit vaccine-induced effector T cells were able to kill up to 90% of the adoptively transferred antigenic peptide-loaded target cell. These results demonstrate that the reduction sensitive NP subunit vaccine elicits a potent CTL response and provide compelling evidence that this approach could be utilized to engineer particulate vaccines to deliver tumor or pathogen associated antigenic peptides to harness the immune system to fight against cancer and infectious diseases.

Pulmonary Delivery of Butyrylcholinesterase as a Model Protein to the Lung.

Pulmonary delivery has great potential for delivering biologics to the lung if the challenges of maintaining activity, stability, and ideal aerosol characteristics can be overcome. To study the interactions of a biologic in the lung, we chose butyrylcholinesterase (BuChE) as our model enzyme, which has application for use as a bioscavenger protecting against organophosphate exposure or for use with pseudocholinesterase deficient patients. In mice, orotracheal administration of free BuChE resulted in 72 h detection in the lungs and 48 h in the broncheoalveolar lavage fluid (BALF). Free BuChE administered to the lung of all mouse backgrounds (Nude, C57BL/6, and BALB/c) showed evidence of an acute cytokine (IL-6, TNF-α, MIP2, and KC) and cellular immune response that subsided within 48 h, indicating relatively safe administration of this non-native biologic. We then developed a formulation of BuChE using Particle Replication in Non-Wetting Templates (PRINT). Aerosol characterization demonstrated biologically active BuChE 1 µm cylindrical particles with a mass median aerodynamic diameter of 2.77 µm, indicative of promising airway deposition via dry powder inhalers (DPI). Furthermore, particulate BuChE delivered via dry powder insufflation showed residence time of 48 h in the lungs and BALF. The in vivo residence time, immune response, and safety of particulate BuChE delivered via a pulmonary route, along with the cascade impaction distribution of dry powder PRINT BuChE, showed promise in the ability to deliver active enzymes with ideal deposition characteristics. These findings provide evidence for the feasibility of optimizing the use of BuChE in the clinic; PRINT BuChE particles can be readily formulated for use in DPIs, providing a convenient and effective treatment option.

Nanoparticle surface charge impacts distribution, uptake and lymph node trafficking by pulmonary antigen-presenting cells.

Engineered nanoparticles have the potential to expand the breadth of pulmonary therapeutics, especially as respiratory vaccines. Notably, cationic nanoparticles have been demonstrated to produce superior local immune responses following pulmonary delivery; however, the cellular mechanisms of this increased response remain unknown. To this end, we investigated the cellular response of lung APCs following pulmonary instillation of anionic and cationic charged nanoparticles. While nanoparticles of both surface charges were capable of trafficking to the draining lymph node and were readily internalized by alveolar macrophages, both CD11b and CD103 lung dendritic cell (DC) subtypes preferentially associated with cationic nanoparticles. Instillation of cationic nanoparticles resulted in the upregulation of Ccl2 and Cxc10, which likely contributes to the recruitment of CD11b DCs to the lung. In total, these cellular mechanisms explain the increased efficacy of cationic formulations as a pulmonary vaccine carrier and provide critical benchmarks in the design of pulmonary vaccine nanoparticles. FROM THE CLINICAL EDITOR: Advance in nanotechnology has allowed the production of precise nanoparticles as vaccines. In this regard, pulmonary delivery has the most potential. In this article, the authors investigated the interaction of nanoparticles with various types of lung antigen presenting cells in an attempt to understand the cellular mechanisms. The findings would further help the future design of much improved vaccines for clinical use.

Subtumoral analysis of PRINT nanoparticle distribution reveals targeting variation based on cellular and particle properties.

The biological activity of nanoparticle-directed therapies critically depends on cellular targeting. We examined the subtumoral fate of Particle Replication in Non-Wetting Templates (PRINT) nanoparticles in a xenografted melanoma tumor model by multi-color flow cytometry and in vivo confocal tumor imaging. These approaches were compared with the typical method of whole-organ quantification by radiolabeling. In contrast to radioactivity based detection which demonstrated a linear dose-dependent accumulation in the organ, flow cytometry revealed that particle association with cancer cells became dose-independent with increased particle doses and that the majority of the nanoparticles in the tumor were associated with cancer cells despite a low fractional association. In vivo imaging demonstrated an inverse relationship between tumor cell association and other immune cells, likely macrophages. Finally, variation in particle size nonuniformly affected subtumoral association. This study demonstrates the importance of subtumoral targeting when assessing nanoparticle activity within tumors. FROM THE CLINICAL EDITOR: Particle Replication in Non-Wetting Templates (PRINT) technology allows the production of nanoparticles with uniform size. The authors in the study utilized PRINT-produced nanoparticles to investigate specific tumor uptake by multi-color flow cytometry and in vivo confocal tumor imaging. This approach allowed further in-depth correlation between nanoparticle properties and tumor cells and should improve future design.

Targeted PRINT Hydrogels: The Role of Nanoparticle Size and Ligand Density on Cell Association, Biodistribution, and Tumor Accumulation.

In this Letter, we varied targeting ligand density of an EGFR binding affibody on the surface of two different hydrogel PRINT nanoparticles (80 nm × 320 and 55 nm × 60 nm) and monitored effects on target-cell association, off-target phagocytic uptake, biodistribution, and tumor accumulation. Interestingly, variations in ligand density only significantly altered in vitro internalization rates for the 80 nm × 320 nm particle. However, in vivo, both particle sizes experienced significant changes in biodistribution and pharmacokinetics as a function of ligand density. Overall, nanoparticle size and passive accumulation were the dominant factors eliciting tumor sequestration.

Rapid and Persistent Delivery of Antigen by Lymph Node Targeting PRINT Nanoparticle Vaccine Carrier To Promote Humoral Immunity.

Nanoparticle delivery of subunit vaccines may increase vaccine efficacy, leading to a wide variety of safe and effective vaccines beyond those available through dosing inactivated or live, attenuated whole pathogens. Here we present a versatile vaccine delivery platform based on PRINT hydrogels made of biocompatible hydroxy-poly(ethylene glycol) (PEG) that is able to activate the complement system by the alternative pathway. These lymph node targeting nanoparticles (NPs) promote the immunogenicity of a model antigen, ovalbumin, showing comparable adjuvant effect to alum. We demonstrate that an antigen-specific humoral response is correlated with antigen delivery to the draining lymph nodes, in particular, B cell rich regions of the lymph nodes. 80 × 180 nm cylindrical NPs were able to sustain prolonged antigen presentation to antigen presenting cells (APCs) and elicit a stronger immune response than nondraining 1 × 1 µm NPs or rapidly clearing soluble antigen. The 80 × 180 nm NPs also show high levels of uptake by key APCs and efficiently stimulate CD4(+) helper T cell proliferation in vivo, further promoting antibody production. These features together produce a significant humoral immune response, superior to that produced by free antigen alone. The simplicity of the chemistries used in antigen conjugation to PRINT NPs confers versatility to this antigen delivery platform, allowing for potential application to many infectious diseases.