Recent advancements in single dose slow-release devices for prophylactic vaccines.

Single dose slow-release vaccines herald a new era in vaccine administration. An ideal device for slow-release vaccine delivery would be minimally invasive and self-administered, making these approaches an attractive alternative for mass vaccination programs, particularly during the time of a pandemic. In this review article, we discuss the latest advances in this field, specifically for prophylactic vaccines able to prevent infectious diseases. Recent studies have found that slow-release vaccines elicit better immune responses and often do not require cold chain transportation and storage, thus drastically reducing the cost, streamlining distribution, and improving efficacy. This promise has attracted significant attention, especially when poor patient compliance of the standard multidose vaccine regimes is considered. Single dose slow-release vaccines are the next generation of vaccine tools that could overcome most of the shortcomings of present vaccination programs and be the next platform technology to combat future pandemics. This article is categorized under: Therapeutic Approaches and Drug Discovery > Emerging Technologies Implantable Materials and Surgical Technologies > Nanomaterials and Implants Biology-Inspired Nanomaterials > Protein and Virus-Based Structures.

Dissolving Microneedle Delivery of a Prophylactic HPV Vaccine.

Prophylactic vaccines capable of preventing human papillomavirus (HPV) infections are still inaccessible to a vast majority of the global population due to their high cost and challenges related to multiple administrations performed in a medical setting. In an effort to improve distribution and administration, we have developed dissolvable microneedles loaded with a thermally stable HPV vaccine candidate consisting of Qß virus-like particles (VLPs) displaying a highly conserved epitope from the L2 protein of HPV (Qß-HPV). Polymeric microneedle delivery of Qß-HPV produces similar amounts of anti-HPV16 L2 IgG antibodies compared to traditional subcutaneous injection while delivering a much smaller amount of intradermal dose. However, a dose sparing effect was found. Furthermore, immunization yielded neutralizing antibody responses in a HPV pseudovirus assay. The vaccine candidate was confirmed to be stable at room temperature after storage for several months, potentially mitigating many of the challenges associated with cold-chain distribution. The ease of self-administration and minimal invasiveness of such microneedle patch vaccines may enable wide-scale distribution of the HPV vaccine and lead to higher patient compliance. The Qß VLP and its delivery technology is a plug-and-play system that could serve as a universal platform with a broad range of applications. Qß VLPs may be stockpiled for conjugation to a wide range of epitopes, which are then packaged and delivered directly to the patient via noninvasive microneedle patches. Such a system paves the way for rapid distribution and self-administration of vaccines.

A Scalable Manufacturing Approach to Single Dose Vaccination against HPV.

Human papillomavirus (HPV) is a globally prevalent sexually-transmitted pathogen, responsible for most cases of cervical cancer. HPV vaccination rates remain suboptimal, partly due to the need for multiple doses, leading to a lack of compliance and incomplete protection. To address the drawbacks of current HPV vaccines, we used a scalable manufacturing process to prepare implantable polymer-protein blends for single-administration with sustained delivery. Peptide epitopes from HPV16 capsid protein L2 were conjugated to the virus-like particles derived from bacteriophage Qß, to enhance their immunogenicity. The HPV-Qß particles were then encapsulated into poly(lactic-co-glycolic acid) (PLGA) implants, using a benchtop melt-processing system. The implants facilitated the slow and sustained release of HPV-Qß particles without the loss of nanoparticle integrity, during high temperature melt processing. Mice vaccinated with the implants generated IgG titers comparable to the traditional soluble injections and achieved protection in a pseudovirus neutralization assay. HPV-Qß implants offer a new vaccination platform; because the melt-processing is so versatile, the technology offers the opportunity for massive upscale into any geometric form factor. Notably, microneedle patches would allow for self-administration in the absence of a healthcare professional, within the developing world. The Qß technology is highly adaptable, allowing the production of vaccine candidates and their delivery devices for multiple strains or types of viruses.

Magnetic Heating Stimulated Cargo Release with Dose Control using Multifunctional MR and Thermosensitive Liposome.

Rationale: Magnetic resonance imaging (MRI) is one of the most widely used diagnostic tools in the clinic. In this setting, real-time monitoring of therapy and tumor site would give the clinicians a handle to observe therapeutic response and to quantify drug amount to optimize the treatment. In this work, we developed a liposome-based cargo (cancer drugs) delivery strategy that could simultaneously monitor the real-time alternating magnetic field-induced cargo release from the change in MRI relaxation parameter R1 and the location and condition of liposome from the change in R2. The tumor site can then be monitored during the cargo release because liposomes would passively target the tumor site through the enhanced permeability and retention (EPR) effect. Physical insights from the experimental results and corresponding Monte Carlo spin dynamics simulations were also discussed. Methods: Superparamagnetic iron oxide (SPIO) nanoparticles, diethylenetriaminepentaacetic acid gadolinium(III) (Gd(III)-DTPA), and a model cancer drug (fluorescein) were co-loaded in PEGylated thermosensitive liposomes. The liposomes were characterized by transmission electron cryo-microscopy (cryoTEM), dynamic light scattering (DLS), and inductively coupled plasma optical emission spectrometry (ICP-OES). Alternating magnetic field (AMF) was used to create controlled mild hyperthermia (39-42°C) and facilitate controlled cargo (fluorescein) release from the thermosensitive liposomes. MRI relaxation parameters, R1 and R2, were measured at room temperature. The temporal variation in R1 was used to obtain the temporal profile of cargo release. Due to their similar sizes, both the gadolinium and cargo (model cancer drug fluorescein) would come out of the liposomes together as a result of heating. The temporal variation in R2 was used to monitor SPIO nanoparticles to enhance the tumor contrast. Monte Carlo spin dynamics simulations were performed by solving the Bloch equations and modeling SPIO nanoparticles as magnetized impenetrable spheres. Results: TEM images and DLS measurements showed the diameter of the liposome nanoparticle ~ 200 nm. AMF heating showed effective release of the model drug. It was found that R1 increased linearly by about 70% and then saturated as the cargo release process was completed, while R2 remained approximately constant with an initial 7%-drop and then recovered. The linear increase in R1 is consistent with the expected linear cargo release with time upon AMF heating. Monte Carlo spin dynamics simulations suggest that the initial temporal fluctuation of R2 is due to the plausible changes of SPIO aggregation and the slow non-recoverable degradation of liposomal membrane that increases water permeability with time by the heating process. The simulations show an order of magnitude increase in R2 at higher water permeability. Conclusion: We have performed MR parameter study of the release of a cargo (model cancer drug, fluorescein) by magnetic heating from thermosensitive multifunctional liposomes loaded with dual contrast agents. The size of the liposome nanoparticles loaded with model cancer drug (fluorescein), gadolinium chelate, and SPIO nanoparticles was appropriate for a variety of cancer therapies. A careful and detailed analysis with theoretical explanation and simulation was carried out to investigate the correlation between MRI relaxation parameters, R1 and R2, and different cargo release fractions. We have quantified the cargo release using R1, which shows a linear relation between each other. This result provides a strong basis for the dosage control of drug delivered. On the other hand, the fairly stable R2 with almost constant value suggests that it could be used to monitor the position and condition of the liposomal site, as SPIO nanoparticles mostly remained in the aqueous core of the liposome. Because our synthesized SPIO-encapsulated liposomes could be targeted to tumor site passively by the EPR effect, or actively through magnetofection, this study provides a solid ground for developing MR cancer theranostics in combination of this nanostructure and AMF heating strategy. Furthermore, our simulation results predict a sharp increase in R2 during the AMF heating, which opens up the exciting possibility of high-resolution, high-contrast real-time imaging of the liposomal site during the drug release process, provided AMF heating could be incorporated into an MRI setup. Our use of the clinically approved materials, along with confirmation by theoretical simulations, make this technique a promising candidate for translational MR cancer theranostics.

Dendrimer- and copolymer-based nanoparticles for magnetic resonance cancer theranostics.

Cancer theranostics is one of the most important approaches for detecting and treating patients at an early stage. To develop such a technique, accurate detection, specific targeting, and controlled delivery are the key components. Various kinds of nanoparticles have been proposed and demonstrated as potential nanovehicles for cancer theranostics. Among them, polymer-like dendrimers and copolymer-based core-shell nanoparticles could potentially be the best possible choices. At present, magnetic resonance imaging (MRI) is widely used for clinical purposes and is generally considered the most convenient and noninvasive imaging modality. Superparamagnetic iron oxide (SPIO) and gadolinium (Gd)-based dendrimers are the major nanostructures that are currently being investigated as nanovehicles for cancer theranostics using MRI. These structures are capable of specific targeting of tumors as well as controlled drug or gene delivery to tumor sites using pH, temperature, or alternating magnetic field (AMF)-controlled mechanisms. Recently, Gd-based pseudo-porous polymer-dendrimer supramolecular nanoparticles have shown 4-fold higher T1 relaxivity along with highly efficient AMF-guided drug release properties. Core-shell copolymer-based nanovehicles are an equally attractive alternative for designing contrast agents and for delivering anti-cancer drugs. Various copolymer materials could be used as core and shell components to provide biostability, modifiable surface properties, and even adjustable imaging contrast enhancement. Recent advances and challenges in MRI cancer theranostics using dendrimer- and copolymer-based nanovehicles have been summarized in this review article, along with new unpublished research results from our laboratories.

Multiple unfolding pathways of leucine binding protein (LBP) probed by single-molecule force spectroscopy (SMFS).

Experimental studies on the folding and unfolding of large multi-domain proteins are challenging, given the proteins' complex energy landscapes with multiple intermediates. Here, we report a mechanical unfolding study of a 346-residue, two-domain leucine binding protein (LBP) from the bacterial periplasm. Forced unfolding of LBP is a prerequisite for its translocation across the cytoplasmic membrane into the bacterial periplasm. During the mechanical stretching of LBP, we observe that 38% of the unfolding flux followed a two-state pathway, giving rise to a single unfolding force peak at ~70 pN with an unfolding contour length of 120 nm in constant-velocity single-molecule pulling experiments. The remaining 62% of the unfolding flux followed multiple three-state pathways, with intermediates having unfolding contour lengths in the range ~20-85 nm. These results suggest that the energy landscape of LBP is complex, with multiple intermediate states, and a large fraction of molecules go through intermediate states during the unfolding process. Furthermore, the presence of the ligand leucine increased the unfolding flux through the two-state pathway from 38% to 65%, indicating the influence of ligand binding on the energy landscape. This study suggests that unfolding through parallel pathways might be a general mechanism for the large two-domain proteins that are translocated to the bacterial periplasmic space.