Repair mechanism of Yishen Tongluo formula on mouse sperm DNA fragmentation caused by polystyrene microplastics.

CONTEXT: Plastics can break down into millions of microplastic (MPs, < 5 mm) particles in the soil and ocean. These MPs can then affect the function of the reproductive system. There is currently no effective solution to this problem aside from traditional Chinese medicine. We have previously used Yishen Tongluo formula (YSTL) to treat sperm DNA damage caused by some toxic substances. OBJECTIVE: To investigate the mechanism underlying the repair of mouse sperm DNA fragmentation caused by polystyrene microplastics by YSTL. MATERIALS AND METHODS: An animal model of polystyrene microplastic (PS-MP)-induced sperm DNA damage was replicated by gavage of SPF ICR (CD1) mice PS-MPs at 1 mg/d and treated with YSTL at 11.89, 23.78 and 47.56 g/kg, respectively, for 60 days. The Sperm DNA fragmentation index (DFI) of each group was detected and compared. The target genes of YSTL identified by transcriptomic and proteomic analyses were validated by qRT-PCR and western blotting. RESULTS: The DFI of the PS group (20.66%) was significantly higher than that of the control group (4.23%). The medium and high doses of the YSTL group (12.8% and 11.31%) exhibited a significant repairing effect. The most enriched pathway was PI3K/Akt. TBL1X, SPARC, hnRNP0, Map7D1, Eps8 and Mrpl27 were screened and SPARC was validated. DISCUSSION AND CONCLUSIONS: The precise mechanism by which YSTL inhibits PD-MPs DNA damage may be associated with the PI3K/Akt pathway and SPARC. It provides a new direction for using traditional Chinese medicine to prevent and repair reproductive system injury caused by MPs.

Alum as an adjuvant for nanoparticle based vaccines: A case study with a hybrid nanoparticle-based nicotine vaccine.

The treatment efficacy of a nicotine vaccine largely relies on its ability to induce high titers of nicotine-specific antibodies. Due to its strong immune-potentiating effects, aluminum salt (Alum) has been commonly used as an adjuvant in various nicotine vaccine formulations. In this study, we attempted to improve the immunological performance of a hybrid nanoparticle-based nicotine vaccine (NanoNicVac) by co-administering it with Alum. It was found that Alum severely restricted the release of NanoNicVac at the site of injection. Moreover, Alum damaged the hybrid structure of the vaccine. In the animal trial, mice immunized with NanoNicVac alone achieved an anti-nicotine IgG titer of 3.5 ±â€¯0.2 × 104 after three injections. Unexpectedly, Alum with quantities of 125, 250, 500, and 1000 µg did not enhance the immunogenicity of NanoNicVac. In addition, Alum did not improve the ability of the vaccine to reduce the entry of nicotine into the brain.

A Nano-in-Nano Polymer-Dendrimer Nanoparticle-Based Nanosystem for Controlled Multidrug Delivery.

Codelivery of multiple chemotherapeutics with different action mechanisms is a promising strategy for cancer treatment. In this study, we developed a novel polymer-dendrimer hybrid nanoparticle-based nanosystem for efficient and controlled codelivery of two model chemotherapeutics, doxorubicin (DOX) and paclitaxel (PTX). The nanosystem was characterized to have a nano-in-nano structure with a size of around 150 nm. The model drugs could feasibly be loaded into the nanosystem ratiometrically with high drug-loading contents by controlling the feeding drug ratios. Also, the model drugs could be released from the nanosystem following a sequential release manner-specifically, quick PTX release and sustained DOX release. Acidic pH was found to enhance the release of both drugs. Moreover, the nanosystem was taken up by cancer cells rapidly and efficiently, and the delivered drugs could release sustainably and efficiently in cells to reach their action targets. In vitro cytotoxicity results demonstrated that, by optimizing drug ratios, the dual-drug-loaded nanosystem could result in better antitumor efficacy than the single-drug-loaded nanosystem or free dual-drug combination. Furthermore, the dual-drug-loaded nanosystem could induce significant changes in both the nucleus and tubulin patterns synergistically. All data suggest that the nano-in-nano polymer-dendrimer hybrid nanoparticle-based nanosystem is a promising candidate to achieve controlled multidrug delivery for effective combination cancer therapy.

Microplastics May Be a Significant Cause of Male Infertility.

Due to the problematic degradation properties of plastics, the decomposition of plastic results in the formation of numerous microplastics (MPs), less than 5 mm in diameter. These MPs enter the soil and the ocean, eventually passing through the air, water, or food chain back to the human body and harming human health. In the last 80 years, male semen analysis parameters have shown a significant decline for unknown reasons, speculated to be caused by pollutants. No studies examined the relationship between human MP exposure and male infertility. In this article, we reviewed the relevant animal experimental research literature in recent years and calculated that the minimum human equivalent dose of MPs leading to abnormal male semen quality is 0.016 mg/kg/d. The literature comparison found that MP exposure in Japan and South Korea was close to this value. These results suggest that MPs can affect male semen quality and that MPs may significantly impact male fertility.

Clinical study on the treatment of male infertility with Wuwei Fuzheng Yijing decoction based on microplastics: Study protocol for a randomized controlled trial.

BACKGROUND: Environmental pollution and male infertility have become global public health problems. The presence of microplastics (MPs) has been detected in the human body, and it has also been demonstrated that MPs can cause damage to the reproductive system. Wuwei Fuzheng Yijing Decoction (WWFZYJ) is effective in treating male infertility. Therefore, we designed a clinical randomized controlled trial to observe the effect of WWFZYJ on the content of MPs and semen quality in male infertility patients, and to evaluate its security. METHODS: In this randomized controlled study, 66 eligible patients were randomly assigned in a 1:1 ratio to a treatment group (WWFZYJ Decoction) and a control group (Coenzyme Q10 tablets combined with vitamin E soft capsules) for 8 weeks. The content of MPs in semen, sperm DNA Fragmentation Index (DFI), and semen analysis (including sperm density, sperm count, forward motile sperm, sperm motility, etc) will be used as primary indicators, and Traditional Chinese Medicine (TCM) syndrome scores will be used as secondary indicators. Vital signs (such as respiration, heart rate, body temperature, blood pressure, electrocardiogram, etc), blood routine, urine routine, stool routine, liver function, and renal function will be used as safety indicators. The primary and secondary indicators will be performed at 0th and 8th week, and the safety indicators will be performed at 0th, 4th, and 8th week. DISCUSSION: This study will provide evidence for the efficacy and safety of WWFZYJ in treating male infertility and reducing the content of MPs in semen, and further explore the effects of MPs on male fertility.

Assembly of single-walled carbon nanohorn supported liposome particles.

Nanoparticle-supported liposomes can be a promising platform for drug delivery, vaccine development, and biomedical imaging. Single-walled carbon nanohorns are a relatively new carbon nanomaterial, and they could be used as carriers of drug and imaging reagents. Assembling lipids around carbon nanohorns would confer this nanomaterial much broader applications such as vaccine development and targeted drug delivery by embedding a target protein or immunogenic protein into the lipid bilayer structure. Here, we show the assembly of functionalized single-walled carbon nanohorns (-CH(2)-CH(2)-COOH(x), ~100 nm) with positively charged lipids through a freeze and thaw cycle. The assembled complex particles can be readily separated from individual nanohorns or liposomes under specific centrifugation conditions. The results from transmission electronic microscopy, flow cytometry through nitrobenzoxadiazole labeled lipids, and zeta potential analysis clearly show that the nanohorns are encapsulated by liposomes with a median size of ca. 120 nm.

Fusion of a family 9 cellulose-binding module improves catalytic potential of Clostridium thermocellum cellodextrin phosphorylase on insoluble cellulose.

Clostridium thermocellum cellodextrin phosphorylase (CtCDP), a single-module protein without an apparent carbohydrate-binding module, has reported activities on soluble cellodextrin with a degree of polymerization (DP) from two to five. In this study, CtCDP was first discovered to have weak activities on weakly water-soluble celloheptaose and insoluble regenerated amorphous cellulose (RAC). To enhance its activity on solid cellulosic materials, four cellulose binding modules, e.g., CBM3 (type A) from C. thermocellum CbhA, CBM4-2 (type B) from Rhodothermus marinus Xyn10A, CBM6 (type B) from Cellvibrio mixtus Cel5B, and CBM9-2 (type C) from Thermotoga maritima Xyn10A, were fused to the C terminus of CtCDP. Fusion of any selected CBM with CtCDP did not influence its kinetic parameters on cellobiose but affected the binding and catalytic properties on celloheptaose and RAC differently. Among them, addition of CBM9 to CtCDP resulted in a 2.7-fold increase of catalytic efficiency for degrading celloheptaose. CtCDP-CBM9 exhibited enhanced specific activities over 20% on the short-chain RAC (DP = 14) and more than 50% on the long-chain RAC (DP = 164). The chimeric protein CtCDP-CBM9 would be the first step to construct a cellulose phosphorylase for in vitro hydrogen production from cellulose by synthetic pathway biotransformation (SyPaB).

Formulation of Nanovaccines toward an Extended Immunity against Nicotine.

Nicotine vaccines have been investigated to assist with smoking cessation. Because smoking cessation is a long process, past nicotine vaccines required multiple injections to achieve long-term efficacy. It would be of great significance if extended efficacy can be achieved with fewer injections. Here, we report the assembly of lipid-polylactic acid (PLA) and lipid-poly(lactic-co-glycolic acid) (PLGA) hybrid nanoparticle (NP) based nicotine vaccines. Mice immunized with the lipid-PLGA vaccine produced higher titers of nicotine-specific antibodies than the lipid-PLA vaccine in short-term. However, the lipid-PLA vaccine was found to induce long-lasting antibodies. Three months after the immunization, only mice that received first two injections of the lipid-PLGA vaccine and a third injection of the lipid-PLA vaccine achieved a significantly lower brain nicotine concentration of 65.13 ± 20.59 ng/mg than 115.88 ± 37.62 ng/mg from the negative controls. The results indicate that not only the stability of the vaccines but also the combination of the vaccines impacted the long-term efficacy of the immunization. Lastly, both the body weight and the histopathology study suggest that the vaccines were safe to mice. These findings suggest that long-term immunity against nicotine can be realized by a rational administration of nanovaccines of different levels of stability.

Tuning the Size of Poly(lactic-co-glycolic Acid) (PLGA) Nanoparticles Fabricated by Nanoprecipitation.

Polymeric nanoparticles (PNPs) are promising drug carriers in cancer treatment. Size of the particles has a significant impact on drug loading, in vivo distribution, extravasation, intratumor diffusion and cell uptake, and thus is critical for the successful development of a drug delivery regime. However, methods for manufacturing PNPs of defined size are yet to be established. The goal of this study is to establish a method that can be used to fabricate PNPs with controlled size. The factors that could impact the size of PNPs fabricated by nano-precipitation are systematically investigated. The factors studied include polymer concentration, organic solvent, temperature, aqueous phase ionic strength, organic phase injection rate, aqueous phase agitation rate, gauge of the needles, and final polymer concentration. Polymer concentration, the choice of organic solvent, temperature, and the ionic strength of the aqueous phase are shown to have a significant impact on the size of PNPs, and the effect of these factors can be attributed to a single parameter, the diffusion coefficient of the solvent in water, Dpw . It is possible that by tightly control these four parameters, nanoparticles with highly predictable and desirable size with narrow size distribution can be fabricated.

Paradox of PEGylation in fabricating hybrid nanoparticle-based nicotine vaccines.

Polyethylene glycol (PEG) has long been used in nanoparticle-based drug or vaccine delivery platforms. In this study, nano-nicotine vaccines (NanoNicVac) were PEGylated to different degrees to investigate the impact of PEG on the immunological efficacy of the vaccine. Hybrid nanoparticles with various degrees of PEGylation (2.5%-30%) were assembled. It was found that 30% PEGylation resulted in a hybrid nanoparticle of a compromised core-shell structure. A higher concentration of PEG also led to a slower cellular uptake of hybrid nanoparticles by dendritic cells. However, increasing the quantity of the PEG could effectively reduce nanoparticle aggregation during storage and improve the stability of the hybrid nanoparticles. Subsequently, nicotine vaccines were synthesized by conjugating nicotine haptens to the differently PEGylated hybrid nanoparticles. In both in vitro and in vivo studies, it was found that a nicotine vaccine with 20% PEGylation (NanoNicVac 20.0) was significantly more stable than the vaccines with lower PEGylation. In addition, NanoNicVac 20.0 induced a significantly higher anti-nicotine antibody titer of 3.7 ±â€¯0.6 × 104 in mice than the other NanoNicVacs with lower concentrations of PEG. In a subsequent pharmacokinetic study, the lowest brain nicotine concentration of 34 ±â€¯11 ng/g was detected in mice that were immunized with NanoNicVac 20.0. In addition, no apparent adverse events were observed in mice immunized with NanoNicVac. In summary, 20% PEGylation confers NanoNicVac with desirable safety, the highest stability, and the best immunological efficacy in mice.

Rationalization of a nanoparticle-based nicotine nanovaccine as an effective next-generation nicotine vaccine: A focus on hapten localization.

A lipid-polymeric hybrid nanoparticle-based next-generation nicotine nanovaccine was rationalized in this study to combat nicotine addiction. A series of nanovaccines, which had nicotine-haptens localized on carrier protein (LPKN), nanoparticle surface (LPNK), or both (LPNKN), were designed to study the impact of hapten localization on their immunological efficacy. All three nanovaccines were efficiently taken up and processed by dendritic cells. LPNKN induced a significantly higher immunogenicity against nicotine and a significantly lower anti-carrier protein antibody level compared to LPKN and LPNK. Meanwhile, it was found that the anti-nicotine antibodies elicited by LPKN and LPNKN bind nicotine stronger than those elicited by LPKN, and LPNK and LPNKN resulted in a more balanced Th1-Th2 immunity than LPKN. Moreover, LPNKN exhibited the best ability to block nicotine from entering the brain of mice. Collectively, the results demonstrated that the immunological efficacy of the hybrid nanoparticle-based nicotine vaccine could be enhanced by modulating hapten localization, providing a promising strategy to combatting nicotine addiction.

The impact of sphingosine kinase inhibitor-loaded nanoparticles on bioelectrical and biomechanical properties of cancer cells.

Cancer progression and physiological changes within the cells are accompanied by alterations in the biophysical properties. Therefore, the cell biophysical properties can serve as promising markers for cancer detection and physiological activities. To aid in the investigation of the biophysical markers of cells, a microfluidic chip has been developed which consists of a constriction channel and embedded microelectrodes. Single-cell impedance magnitudes at four frequencies and entry and travel times are measured simultaneously during their transit through the constriction channel. This microchip provides a high-throughput, label-free, automated assay to identify biophysical signatures of malignant cells and monitor the therapeutic efficacy of drugs. Here, we monitored the dynamic cellular biophysical properties in response to sphingosine kinase inhibitors (SphKIs), and compared the effectiveness of drug delivery using poly lactic-co-glycolic acid (PLGA) nanoparticles (NPs) loaded with SphKIs versus conventional delivery. Cells treated with SphKIs showed significantly higher impedance magnitudes at all four frequencies. The bioelectrical parameters extracted using a model also revealed that the highly aggressive breast cells treated with SphKIs shifted electrically towards that of a less malignant phenotype; SphKI-treated cells exhibited an increase in cell-channel interface resistance and a significant decrease in specific membrane capacitance. Furthermore, SphKI-treated cells became slightly more deformable as measured by a decrease in their channel entry and travel times. We observed no significant difference in the bioelectrical changes produced by SphKI delivered conventionally or with NPs. However, NPs-packaged delivery of SphKI decreased the cell deformability. In summary, this study showed that while the bioelectrical properties of the cells were dominantly affected by SphKIs, the biomechanical properties were mainly changed by the NPs.

Engineering the lipid layer of lipid-PLGA hybrid nanoparticles for enhanced in vitro cellular uptake and improved stability.

Lipid-polymer hybrid nanoparticles (NPs), consisting of a polymeric core and a lipid shell, have been intensively examined as delivery systems for cancer drugs, imaging agents, and vaccines. For applications in vaccine particularly, the hybrid NPs need to be able to protect the enclosed antigens during circulation, easily be up-taken by dendritic cells, and possess good stability for prolonged storage. However, the influence of lipid composition on the performance of hybrid NPs has not been well studied. In this study, we demonstrate that higher concentrations of cholesterol in the lipid layer enable slower and more controlled antigen release from lipid-poly(lactide-co-glycolide) acid (lipid-PLGA) NPs in human serum and phosphate buffered saline (PBS). Higher concentrations of cholesterol also promoted in vitro cellular uptake of hybrid NPs, improved the stability of the lipid layer, and protected the integrity of the hybrid structure during long-term storage. However, stabilized hybrid structures of high cholesterol content tended to fuse with each other during storage, resulting in significant size increase and lowered cellular uptake. Additional experiments demonstrated that PEGylation of NPs could effectively minimize fusion-caused size increase after long term storage, leading to improved cellular uptake, although excessive PEGylation will not be beneficial and led to reduced improvement. STATEMENT OF SIGNIFICANCE: This paper reports the engineering of the lipid layer that encloses a polymeric nanoparticle, which can be used as a carrier for drug and vaccine molecules for targeted delivery. We demonstrated that the concentration of cholesterol is critical for the stability and uptake of the hybrid nanoparticles by dendritic cells, a targeted cell for the delivery of immune effector molecules. However, we found that hybrid nanoparticles with high cholesterol concentration tend to fuse during storage resulting in larger particles with decreased cellular uptake. This problem is subsequently solved by PEGylating the hybrid nanoparticles. With increased research and clinical applications of lipid-polymer hybrid nanoparticles in drug and vaccine delivery, this work will significantly impact the design of the hybrid nanoparticles for minimized molecule release during circulation and increased bioavailability of the target molecules.

Negatively Charged Carbon Nanohorn Supported Cationic Liposome Nanoparticles: A Novel Delivery Vehicle for Anti-Nicotine Vaccine.

Tobacco addiction is the second-leading cause of death in the world. Due to the nature of nicotine (a small molecule), finding ways to combat nicotine's deleterious effects has been a constant challenge to the society and the medical field. In the present work, a novel anti-nicotine vaccine based on nanohorn supported liposome nanoparticles (NsL NPs) was developed. The nano-vaccine was constructed by using negatively charged carbon nanohorns as a scaffold for the assembly of cationic liposomes, which allow the conjugation of hapten conjugated carrier proteins. The assembled bio-nanoparticles are stable. Mice were immunized subcutaneously with the nano-vaccine, which induced high titer and high affinity of nicotine specific antibodies in mice. Furthermore, no evidence of clinical signs or systemic toxicity followed multiple administrations of NsL-based anti-nicotine vaccine. These results suggest that NsL-based anti-nicotine vaccine is a promising candidate in treating nicotine dependence and could have potential to significantly contribute to smoking cessation.

Tobacco protein separation by aqueous two-phase extraction.

Tobacco has long been considered as a host to produce large quantity of high-valued recombinant proteins. However, dealing with large quantities of biomass is a challenge for downstream processing. Aqueous two-phase extraction (ATPE) has been widely used in purifying proteins from various sources. It is a protein-friendly process and can be scaled up easily. In this paper, ATPE was studied for its applicability to recombinant protein purification from tobacco with egg white lysozyme as the model protein. Separate experiments with poly(ethylene glycol) (PEG)-salt-tobacco extract and PEG-salt-lysozyme were carried out to determine the partition behavior of tobacco protein and lysozyme, respectively. Two-level fractional factorial designs were used to study the effects of factors such as, PEG molecular mass, PEG concentration, the concentration of phase forming salt, sodium chloride concentration and pH, on protein partitioning. The results showed that, among the studied systems, PEG-sodium sulfate system was most suitable for lysozyme purification. Detailed experiments were conducted by spiking lysozyme into the tobacco extract. The conditions with highest selectivity of lysozyme over native tobacco protein were determined using a response surface design. The purification factor was further improved by decreasing the phase ratio along the tie line corresponding to the phase compositions with the highest selectivity. Under selected conditions the lysozyme yield was predicted to be 87% with a purification factor of 4 and concentration factor of 14. From this study, ATPE was shown to be suitable for initial protein recovery and partial purification from transgenic tobacco.

A novel and efficient nicotine vaccine using nano-lipoplex as a delivery vehicle.

A number of vaccines conjugating nicotine haptens with carrier proteins have been developed to combat nicotine caused tobacco dependence. Some vaccines, such as NicVAX, NicQb, advanced into clinical trials, but none of them were successful. Most of those vaccines have some innate disadvantages such as low nicotine loading capacity, easy degradation, and vulnerable to the clearance by reticulo-endothelial system (RES). Thus, there is undoubtedly an urgent need for developing novel vaccines against nicotine addiction. In this study, we assembled a liposome-protein based nanoparticle as a nicotine hapten delivery system. The nanoparticle (Scheme 1) was constructed by conjugating a model hapten carrier protein, bovine serum albumin (BSA), to cationic liposomes. This nano-sized complex, lipoplex, was characterized using zetasizer, transmission electron microscope (TEM), and flow cytometry. The efficacy of the lipoplex vaccine was evaluated in mice and compared with that of Nicotine-BSA conjugate (Nic-BSA). The lipoplex vaccine with Alum was able to elicit the highest NicAb titer of 11169±2112, which was significantly higher than that induced by either the vaccine without Alum or Nic-BSA with Alum. The significant immunostimulatory effect of this nano-lipoplex may provide a novel strategy to improve the immunogenic ability of current nicotine vaccines or other vaccines using small molecules as immunogens.

Assembly of bio-nanoparticles for double controlled drug release.

A critical limiting factor of chemotherapy is the unacceptably high toxicity. The use of nanoparticle based drug carriers has significantly reduced the side effects and facilitated the delivery of drugs. Source of the remaining side effect includes (1) the broad final in vivo distribution of the administrated nanoparticles, and (2) strong basal drug release from nanoparticles before they could reach the tumor. Despite the advances in pH-triggered release, undesirable basal drug release has been a constant challenge under in vivo conditions. In this study, functionalized single walled carbon nanohorn supported immunoliposomes were assembled for paclitaxel delivery. The immunoliposomes were formulated with polyethylene glycol, thermal stable and pH sensitive phospholipids. Each nanohorn was found to be encapsulated within one immunoliposome. Results showed a highly pH dependent release of paclitaxel in the presence of serum at body temperature with minimal basal release under physiological conditions. Upon acidification, paclitaxel was released at a steady rate over 30 days with a cumulative release of 90% of the loaded drug. The drug release results proved our hypothesized double controlled release mechanism from the nanoparticles. Other results showed the nanoparticles have doubled loading capacity compared to that of traditional liposomes and higher affinity to breast cancer cells overexpressing Her2 receptors. Internalized nanoparticles were found in lysosomes.

A salt-bridge controlled by ligand binding modulates the hydrolysis reaction in a GH5 endoglucanase.

Cellulases, distributed in at least 15 families of glycoside hydrolases, will play a key role in biomass conversion and renewable energy challenges of the future. Cel5B from Clostridium thermocellum is a ß-1,4-endoglucanase and a member of family 5 of glycoside hydrolases (GH5) and is characterized by an (α/ß)(8) barrel structure. In contrast to other retaining enzymes, in which the catalytic carboxylate groups (glutamate or aspartate) are positioned ≈ 5.5 Å apart to facilitate nucleophilic attack on the anomeric carbon of the sugar substrate, these two residues in Cel5B are positioned ≈ 10 Å from each other in the unliganded wild-type structure. The structure of the enzyme solved in complex with a cleavage product (cellobiose) revealed ligand-induced conformational changes in the loop carrying Glu140 (proton donor). The reorientation of Glu140 in the complex reduces the separation of the catalytic glutamate residues to 4.3 Å. In this study, we took advantage of conventional and steered molecular dynamics (MD) simulations along with in silico and in vitro mutagenesis to investigate the ligand-induced changes of the enzyme and interactions involved in preservation of Cel5B conformations in the presence and absence of substrate. We determined that the variation in separation of catalytic glutamates in the absence and presence of substrate is due to the different protonation states of the proton donor glutamate that is largely governed by conformational changes in the ß3α3 loop. In the absence of substrate, the conformation of Cel5B is preserved by an electrostatic interaction between deprotonated Glu140 and protonated His91. The ion pair is interrupted upon the binding of substrate, and the positional displacement of the ß3α3 loop allows Glu140 to become oriented within the active site in a less hydrophilic microenvironment that assists in Glu140 protonation.

Assembly and characterization of lipid-lipid binding protein particles.

Lipid-protein complexes, lipoplexes, are currently of great interest because of their immunogenic, gene free, and low cost properties. For their applications as potential vaccines, it is critical to display a target protein on the surface of lipoplex particles to allow external interactions to take place. However, how to effectively assemble lipoplexes with target proteins externally accessible is a constant challenge. In this study, human liver fatty acid binding protein 1 (hl-FABP1) was used as a model protein in lipoplex assembly with a non-lipid binding protein, bovine serum albumin (BSA), serving as a comparison. The protein-lipid particles were assembled by four different processes and characterized by dynamic light scattering (DLS), transmission electron microscope (TEM), flow cytometry (FCM), and a modified ELISA. Results indicate that by incubating the target protein with pre-formed liposomes at a temperature higher than all transition temperatures (T(m)) of the lipids used through an extended period of time, 1.48×10(-6)nmol per lipoplex of incorporated proteins can be detected by ELISA and are externally accessible. Additional experiments showed that most of those externally accessible proteins are likely embedded in the lipid bilayer structure and are not subject to dissociation from the lipoplex particles at elevated salt concentrations.

The immunological and pharmacokinetic evaluation of Lipid-PLGA hybrid nanoparticle-based oxycodone vaccines.

The current opioid epidemic is one of the most profound public health crises facing the United States. Despite that it has been under the spotlight for years, available treatments for opioid use disorder (OUD) and overdose are limited to opioid receptor ligands such as the agonist methadone and the overdose reversing drugs such as naloxone. Vaccines are emerging as an alternative strategy to combat OUD and prevent relapse and overdose. Most vaccine candidates consist of a conjugate structure containing the target opioid attached to an immunogenic carrier protein. However, conjugate vaccines have demonstrated some intrinsic shortfalls, such as fast degradation and poor recognition by immune cells. To overcome these challenges, we proposed a lipid-PLGA hybrid nanoparticle (hNP)-based vaccine against oxycodone (OXY), which is one of the most frequently misused opioid analgesics. The hNP-based OXY vaccine exhibited superior immunogenicity and pharmacokinetic efficacy in comparison to its conjugate vaccine counterpart. Specifically, the hNP-based OXY vaccine formulated with subunit keyhole limpet hemocyanin (sKLH) as the carrier protein and aluminum hydroxide (Alum) as the adjuvant (OXY-sKLH-hNP(Alum)) elicited the most potent OXY-specific antibody response in mice. The induced antibodies efficiently bound with OXY molecules in blood and suppressed their entry into the brain. In a following dose-response study, OXY-sKLH-hNP(Alum) equivalent to 60 µg of sKLH was determined to be the most promising OXY vaccine candidate moving forward. This study provides evidence that hybrid nanoparticle-based vaccines may be superior vaccine candidates than conjugate vaccines and will be beneficial in treating those suffering from OUD.