BackgroundWe previously developed a saline-formulated mixture of 4 synthetic messenger ribonucleic acids (mRNAs) encoding the cytokines IL-12, IFN-α, GM-CSF, and IL-15 sushi (IL-15-IL-15Rα receptor ...fusion) that promoted systemic immunity and tumor eradication upon intratumoral administration in multiple mouse tumor models.Here, we explored whether stabilization of the mRNAs through a lipoplex (LPX) formulation could confer increased expression of the encoded cytokines to result in improved antitumor efficacy. To this end, we developed a pH-responsive amphoteric LPX by mixing preformed liposomes with mRNA in aqueous/aqueous solutions. The resulting complex was designed to destabilize under neutral pH in circulation, leading to a limited exposure in non-target organs following intratumoral administration.MethodsBiological activity of LPX-formulated mRNA-encoded cytokines or reporter genes were compared to that of naked mRNA in vivo using C57BL/6 and BALB/c mice harboring TC-1, B16F10 and CT26 tumors, respectively. Following intratumoral administration, cytokines were analyzed in serum and organs by ELISA and luciferase expression was followed by bioluminescent imaging. Anti-tumor efficacy was assessed by tumor growth inhibition and survival of CT26-tumor bearing mice and expansion of tumor-reactive T cells were analyzed by flow cytometry. Abscopal effects were assessed in C57BL/6 mice bearing B16F10 subcutaneous tumors on both flanks.ResultsIn vivo studies demonstrated a favorable tumor-to-liver translation ratio of luciferase reporter mRNA formulated with the LPX, supporting the hypothesis that the LPX would destabilize in circulation. The LPX formulation further increased target expression of each of the four encoded cytokines in tumors ranging from 7 to 47-fold compared to saline solution in B16F10-tumor bearing animals. Intratumoral administration of LPX-mRNA to mice with subcutaneous CT26 colorectal tumors significantly inhibited tumor growth, with stronger tumor growth inhibition when compared to treatment with saline-formulated mRNA. This increased antitumor activity was correlated with stronger pharmacodynamic responses, including expansion of T cells reactive against the tumor antigen gp70. C57BL/6 mice bearing B16F10 subcutaneous tumors on both flanks received an intratumoral injection of LPX-mRNA on one side. Treatment resulted in significant inhibition of tumor growth and prolonged survival. Antitumor efficacy was further enhanced when combined with anti-PD-1 antibody, resulting in complete tumor regression in a subset of mice, consistent with an abscopal response.ConclusionsTogether, these results demonstrate the potent anticancer activity of a novel LPX-formulated mRNA mixture.Ethics ApprovalAnimal experiments were performed according to the guidelines of the Institutional Animal Care and Use Committee (IACUC) and in accordance with the regulations of the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC).
A safe and effective vaccine against COVID-19 is urgently needed in quantities that are sufficient to immunize large populations. Here we report the preclinical development of two vaccine candidates ...(BNT162b1 and BNT162b2) that contain nucleoside-modified messenger RNA that encodes immunogens derived from the spike glycoprotein (S) of SARS-CoV-2, formulated in lipid nanoparticles. BNT162b1 encodes a soluble, secreted trimerized receptor-binding domain (known as the RBD-foldon). BNT162b2 encodes the full-length transmembrane S glycoprotein, locked in its prefusion conformation by the substitution of two residues with proline (S(K986P/V987P); hereafter, S(P2) (also known as P2 S)). The flexibly tethered RBDs of the RBD-foldon bind to human ACE2 with high avidity. Approximately 20% of the S(P2) trimers are in the two-RBD 'down', one-RBD 'up' state. In mice, one intramuscular dose of either candidate vaccine elicits a dose-dependent antibody response with high virus-entry inhibition titres and strong T-helper-1 CD4
and IFNγ
CD8
T cell responses. Prime-boost vaccination of rhesus macaques (Macaca mulatta) with the BNT162b candidates elicits SARS-CoV-2-neutralizing geometric mean titres that are 8.2-18.2× that of a panel of SARS-CoV-2-convalescent human sera. The vaccine candidates protect macaques against challenge with SARS-CoV-2; in particular, BNT162b2 protects the lower respiratory tract against the presence of viral RNA and shows no evidence of disease enhancement. Both candidates are being evaluated in phase I trials in Germany and the USA
, and BNT162b2 is being evaluated in an ongoing global phase II/III trial (NCT04380701 and NCT04368728).
Sensors incorporating molecularly imprinted polymers (MIPs) are feasible in concept though the reproducibility of such devices can be compromised by the large number of interdependent steps. For this ...reason, many researchers have focused on the synthesis of MIP particles only, not on their immobilization. Herein is presented a sol–gel based method for immobilization of unmodified MIP particles for use in an electrochemical sensor. The macroporous particles were prepared using precipitation-polymerization and imprinted with theophylline. The sol–gel was combined with graphite microparticles (50 μm) and the composite was deposited on the surfaced of an epoxy-graphite electrode. The sensor was then tested for its response to theophylline using differential pulse voltammetry. A limit of detection of 1 μM was observed and a relative standard deviation of 6.85 %. The electrode can be regenerated via a thermal washing process which was accompanied by an initial signal loss of 29.3 %. Any further regeneration caused a signal loss of 2.4 % only.
Graphical Abstract
A voltammetric sensor for the preferential detection of theophylline is prepared based on molecularly imprinted microspheres immobilized in a sol-gel layer. The use of precipitated polymer microparticles allows for the effective regeneration of the sensor using an acidic wash.
A novel protocol for use of molecularly imprinted polymer (MIP) in analysis of melamine is presented. Design of polymer for melamine has been achieved using a combination of computational techniques ...and laboratory trials, the former greatly reducing the duration of the latter. The compatibility and concerted effect of monomers and solvents were also investigated and discussed. Two novel open-source tools were presented which are the online polymer calculator from mipdatabase.com and the application of the Gromacs modelling suite to determine the ideal stoichiometric ratio between template and functional monomer. The MIP binding was characterised for several structural analogues at 1-100 μM concentrations. The use of divinylbenzene (DVB) as cross-linking polymer and itaconic acid as functional monomer allowed synthesis of MIP with imprint factor (IF) of 2.25 for melamine. This polymer was used in high-performance liquid chromatography (HPLC) for the rapid detection of melamine in spiked milk samples with an experimental run taking 7-8 min. This approach demonstrated the power of virtual tools in accelerated design of MIPs for practical applications.
•First report on MISPE-LC–MS/MS analysis of four cannabinoids in urine and oral fluid.•Screening a NIP-library to optimize the synthesis of MIPs for cannabinoids.•Recoveries ranging from 50.3 to ...81.3% and 64.9 to 111% in urine and OF, respectively.•Sensitive, simple and cost effective methodology for routine analysis in urine.•Identification of recent cannabis consumption in both matrices.
A novel molecularly imprinted solid phase extraction (MISPE) methodology followed by liquid chromatography tandem mass spectrometry (LC–MS/MS) has been developed using cylindrical shaped molecularly imprinted pills for detection of Δ9-tetrahydrocannabinol (THC), 11-nor-Δ9-tetrahydrocannabinol carboxylic acid (THC-COOH), cannabinol (CBN) and cannabidiol (CBD) in urine and oral fluid (OF). The composition of the molecular imprinted polymer (MIP) was optimized based on the screening results of a non-imprinted polymer library (NIP-library). Thus, acrylamide as functional monomer and ethylene glycol dimethacrylate as cross-linker were selected for the preparation of the MIP, using catechin as a mimic template. MISPE pills were incubated with 0.5mL urine or OF sample for adsorption of analytes. For desorption, the pills were transferred to a vial with 2mL of methanol:acetic acid (4:1) and sonicated for 15min. The elution solvent was evaporated and reconstituted in methanol:formic acid (0.1%) 50:50 to inject in LC–MS/MS. The developed method was linear over the range from 1 to 500ngmL−1 in urine and from 0.75 to 500ngmL−1 in OF for all four analytes. Intra- and inter-day imprecision were <15%. Extraction recovery was 50–111%, process efficiency 15.4–54.5% and matrix effect ranged from −78.0 to −6.1%. Finally, the optimized and validated method was applied to 4 urine and 5 OF specimens. This is the first method for the determination of THC, THC-COOH, CBN and CBD in urine and OF using MISPE technology.
Molecularly Imprinted Polymers (MIPs) are highly advantageous in the field of analytical chemistry. However, interference from secondary molecules can also impede capture of a target by a MIP ...receptor. This greatly complicates the design process and often requires extensive laboratory screening which is time consuming, costly, and creates substantial waste products. Herein, is presented a new technique for screening of “virtually imprinted receptors” for rebinding of the molecular template as well as secondary structures, correlating the virtual predictions with experimentally acquired data in three case studies. This novel technique is particularly applicable to the evaluation and prediction of MIP receptor specificity and efficiency in complex aqueous systems.
A novel method for “virtual molecular imprinting” and evaluation of the resulting receptor for docking specificity and affinity: the greater degree of ab initio insight that this technique provides to the scientist regarding receptor affinity and design can greatly reduce the workload and waste production, thus creating a more efficient and greener laboratory.
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