Nanoparticle-delivered TLR4 and RIG-I agonists enhance immune response to SARS-CoV-2 subunit vaccine.

Despite success in vaccinating populations against SARS-CoV-2, concerns about immunity duration, continued efficacy against emerging variants, protection from infection and transmission, and worldwide vaccine availability remain. Molecular adjuvants targeting pattern recognition receptors (PRRs) on antigen-presenting cells (APCs) could improve and broaden the efficacy and durability of vaccine responses. Native SARS-CoV-2 infection stimulates various PRRs, including toll-like receptors (TLRs) and retinoic acid-inducible gene I (RIG-I)-like receptors. We hypothesized that targeting PRRs using molecular adjuvants on nanoparticles (NPs) along with a stabilized spike protein antigen could stimulate broad and efficient immune responses. Adjuvants targeting TLR4 (MPLA), TLR7/8 (R848), TLR9 (CpG), and RIG-I (PUUC) delivered on degradable polymer NPs were combined with the S1 subunit of spike protein and assessed in vitro with isogeneic mixed lymphocyte reactions (isoMLRs). For in vivo studies, the adjuvant-NPs were combined with stabilized spike protein or spike-conjugated NPs and assessed using a two-dose intranasal or intramuscular vaccination model in mice. Combination adjuvant-NPs simultaneously targeting TLR and RIG-I receptors (MPLA+PUUC, CpG+PUUC, and R848+PUUC) differentially induced T cell proliferation and increased proinflammatory cytokine secretion by APCs in vitro. When delivered intranasally, MPLA+PUUC NPs enhanced CD4+CD44+ activated memory T cell responses against spike protein in the lungs while MPLA NPs increased anti-spike IgA in the bronchoalveolar (BAL) fluid and IgG in the blood. Following intramuscular delivery, PUUC NPs induced strong humoral immune responses, characterized by increases in anti-spike IgG in the blood and germinal center B cell populations (GL7+ and BCL6+ B cells) in the draining lymph nodes (dLNs). MPLA+PUUC NPs further boosted spike protein-neutralizing antibody titers and T follicular helper cell populations in the dLNs. These results suggest that protein subunit vaccines with particle-delivered molecular adjuvants targeting TLR4 and RIG-I could lead to robust and unique route-specific adaptive immune responses against SARS-CoV-2.

Amphiphilic mannose-6-phosphate glycopolypeptide-based bioactive and responsive self-assembled nanostructures for controlled and targeted lysosomal cargo delivery.

Receptors of carbohydrate mannose-6-phosphate (M6P) are overexpressed in specific cancer cells (such as breast cancer) and are also involved in the trafficking of mannose-6-phosphate labeled proteins exclusively onto lysosomes via cell surface M6P receptor (CI-MPR) mediated endocytosis. Herein, for the first time, mannose-6-phosphate glycopolypeptide (M6PGP)-based bioactive and stimuli-responsive nanocarriers are reported. They are selectively taken up via receptor-mediated endocytosis, and trafficked to lysosomes where they are subsequently degraded by pH or enzymes, leading to the release of the cargo inside the lysosomes. Two different amphiphilic M6P block copolymers M6PGP15-APPO44 and M6PGP15-(PCL25)2 were synthesized by click reaction of the alkyne end-functionalized M6PGP15 with pH-responsive biocompatible azide end-functionalized acetal PPO and azide end-functionalized branched PCL, respectively. In water, the amphiphilic M6P-glycopolypeptide block copolymers self-assembled into micellar nanostructures, as was evidenced by DLS, TEM, AFM, and fluorescence spectroscopy techniques. These micellar systems were competent to encapsulate the hydrophobic dye rhodamine-B-octadecyl ester, which was used as the model drug. They were stable at physiological pH but were found to disassemble at acidic pH (for M6PGP15-APPO44) or in the presence of esterase (for M6PGP15-(PCL25)2). These M6PGP based micellar nanoparticles can selectively target lysosomes in cancerous cells such as MCF-7 and MDA-MB-231. Finally, we demonstrate the clathrin-mediated endocytic pathway of the native FL-M6PGP polymer and RBOE loaded M6PGP micellar-nanocarriers, and selective trafficking of MCF-7 and MDA-MB-231 breast cancer cell lysosomes, demonstrating their potential applicability toward receptor-mediated lysosomal cargo delivery.

pH-Responsive "Supra-Amphiphilic" Nanoparticles Based on Homoarginine Polypeptides.

pH-responsive "supra-amphiphiles" based on double hydrophilic, positively charged block copolypeptides such as PEG-b-poly-l-lysine together with low molecular weight stimuli-sensitive partners that contain phosphate and carboxylate groups have been widely studied. In contrast, the other widely used cationic polypeptide poly-l-arginine whose cell-penetrating properties are well-known has been much less explored for the synthesis of supra-amphiphile-based nanomaterials. It is also known that the guanidine side chain of arginine binds to carboxylate anions with binding constants that are 2.5 times higher than the corresponding amines of poly-l-lysine. Here, we demonstrate the fabrication of pH-sensitive supra-amphiphilic nanoparticles by simple mixing of PEG2k-b-poly(homoarginine) block copolymer and carboxylic acid containing functional low molecular weight organic compounds. A high yielding three-step methodology was developed for the synthesis of ε-N,N'-di-Boc-l-homoarginine-α-N-carboxyanhydride which was polymerized using amine-terminated PEG (2000 MW) to yield 100% guanine-functionalized polypeptide (PEG2k-b-PHR30) with controlled molecular weights and low PDIs. Incubation of PEG2k-b-PHR30 with four different carboxylic acids (including dexamethasone a glucocorticoid receptor used in cancer therapy) in water leads to the formation of "supra-amphiphilic" nanoparticles (<200 nm size) due to the charge neutralization resulting from the strong interaction between the guanidine group and the carboxylate group. All these nanoparticles were able to encapsulate the hydrophobic dye Nile red with varying efficiency. Although these assemblies were stable at neutral pH, upon lowering the pH of the solution between 4 and 5, the protonation of the carboxylic acids leads to disassembly of these nanoparticles. The cytotoxicity of all four "supra-amphiphilic" nanoparticles varied depending on the carboxylic acid used for their fabrication. While the nanoparticle formed using dioctylbenzoic acid displayed 80% cell viability at concentration of 60 µg/mL, those formed with the steroid deoxycholic acid or dexamethasone showed only 40% cell viability at similar concentrations. Colocalization studies performed using epifluorescence microscopy demonstrate the successful uptake of intact dye-encapsulated nanoparticle inside the cell.

Amphiphilic Glycopolypeptide Star Copolymer-Based Cross-Linked Nanocarriers for Targeted and Dual-Stimuli-Responsive Drug Delivery.

Glycopolypeptide-based nanocarriers are an attractive class of drug delivery vehicles because of the involvement of carbohydrates in the receptor-mediated endocytosis process. To enhance their efficacy toward controlled and programmable drug delivery, we have prepared stable glycopolypeptide-based bioactive dual-stimuli-responsive (redox and enzyme) micelles for delivery of anticancer drugs specifically to the cancer cells. The amphiphilic biocompatible miktoarm star copolymer, which comprises two hydrophobic poly(ε-caprolactone) blocks, a short poly(propargyl glycine) middle block, and a hydrophilic galactose glycopolypeptide block, was designed and synthesized. The star copolymer is initially self-assembled into un-cross-linked (UCL) micelles, and free alkyne groups at the core-shell interface of the UCL micelles, which were cross-linked by bis(azidoethyl) disulfide (BADS) via click chemistry to form interface cross-linked (ICL) micelles. ICL micelles were found to be stable against dilution. BADS imparted redox-responsive properties to the micelles, while PCL rendered them enzyme-degradable. Dual-stimuli-responsive release behavior with Dox as model drug was studied individually as well as synergistically by applying two stimuli in different sequences. The galactose-containing UCL and ICL micelles were shown to be nontoxic. Intracellular Dox release from UCL and ICL micelles was demonstrated in liver cancer cells (HepG2) by time-dependent cellular uptake studies, and controlled release from ICL micelles compared to UCL micelles was observed. The present report opens a new approach toward targeted and programmable drug delivery in tumor tissues via a specifically targeted (receptor-mediated), dual-responsive, and stable cross-linked nanocarrier system.

Nucleophilic versus Electrophilic Reactivity of Bioinspired Superoxido Nickel(II) Complexes.

The formation and detailed spectroscopic characterization of the first biuret-containing monoanionic superoxido-NiII intermediate [LNiO2 ]- as the Li salt [2; L=MeN[C(=O)NAr)2 ; Ar=2,6-iPr2 C6 H3 )] is reported. It results from oxidation of the corresponding [Li(thf)3 ]2 [LNiII Br2 ] complex M with excess H2 O2 in the presence of Et3 N. The [LNiO2 ]- core of 2 shows an unprecedented nucleophilic reactivity in the oxidative deformylation of aldehydes, in stark contrast to the electrophilic character of the previously reported neutral Nacnac-containing superoxido-NiII complex 1, [L'NiO2 ] (L'=CH(CMeNAr)2 ). According to density-functional theory (DFT) calculations, the remarkably different behaviour of 1 versus 2 can be attributed to their different charges and a two-state reactivity, in which a doublet ground state and a nearby spin-polarized doublet excited-state both contribute in 1 but not in 2. The unexpected nucleophilicity of the superoxido-NiII core of 2 suggests that such a reactivity may also play a role in catalytic cycles of Ni-containing oxygenases and oxidases.