Generating long-lived mucosal and systemic antibodies through respiratory immunization with protective antigens encapsulated in nanoscale biodegradable particles could potentially decrease or eliminate the incidence of many infectious diseases, but requires the incorporation of a suitable mucosal immunostimulant. We previously found that respiratory immunization with a model protein antigen (LPS-free OVA) encapsulated in PLGA 50:50 nanoparticles (~380 nm diameter) surface-modified with complement peptide-derived immunostimulant 02 (CPDI-02; formerly EP67) through 2 kDa PEG linkers increases mucosal and systemic OVA-specific memory T-cells with long-lived surface phenotypes in young, naïve female C57BL/6 mice. Here, we determined if respiratory immunization with LPS-free OVA encapsulated in similar PLGA 50:50 microparticles (~1 μm diameter) surface-modified with CPDI-02 (CPDI-02-MP) increases long-term OVA-specific mucosal and systemic antibodies. We found that, compared to MP surface-modified with inactive, scrambled scCPDI-02 (scCPDI-02-MP), intranasal administration of CPDI-02-MP in 50 μL sterile PBS greatly increased titers of short-term (14 days post-immunization) and long-term (90 days post-immunization) antibodies against encapsulated LPS-free OVA in nasal lavage fluids, bronchoalveolar lavage fluids, and sera of young, naïve female C57BL/6 mice with minimal lung inflammation. Thus, surface modification of ~1 μm biodegradable microparticles with CPDI-02 is likely to increase long-term mucosal and systemic antibodies against encapsulated protein antigen after respiratory and possibly other routes of mucosal immunization.

RNA interference (RNAi) molecules have tremendous potential for cancer therapy but are limited by insufficient potency after intravenous (IV) administration. We previously found that polymer complexes (polyplexes) formed between 3′-cholesterol-modified siRNA (Chol-siRNA) or DsiRNA (Chol-DsiRNA) and the cationic diblock copolymer PLL[30]-PEG[5K] greatly increase RNAi potency against stably expressed LUC mRNA in primary syngeneic murine breast tumors after daily IV dosing. Chol-DsiRNA polyplexes, however, maintain LUC mRNA suppression for ~48 h longer after the final dose than Chol-siRNA polyplexes, which suggests that they are the better candidate formulation. Here, we directly compared the activities of Chol-siRNA polyplexes and Chol-DsiRNA polyplexes in primary murine 4T1 breast tumors against STAT3, a therapeutically relevant target gene that is overexpressed in many solid tumors, including breast cancer. We found that Chol-siSTAT3 polyplexes suppressed STAT3 mRNA in 4T1 tumors with similar potency (half-maximal ED₅₀ 0.3 mg/kg) and kinetics (over 96 h) as Chol-DsiSTAT3 polyplexes, but with slightly lower activity against total Stat3 protein (29% vs. 42% suppression) and tumor growth (11.5% vs. 8.6% rate-based T/C ratio) after repeated IV administration of equimolar, tumor-saturating doses every other day. Thus, both Chol-siRNA polyplexes and Chol-DsiRNA polyplexes may be suitable clinical candidates for the RNAi therapy of breast cancer and other solid tumors.

RNA interference (RNAi) molecules have tremendous potential for cancer therapy but are limited by insufficient potency after i.v. administration. We previously found that polymer complexes (polyplexes) formed between 3’-cholesterol-modified siRNA (Chol-siRNA) or DsiRNA (Chol-DsiRNA) and the cationic diblock copolymer PLL[30]-PEG[5K] greatly increase RNAi potency against stably expressed LUC mRNA in primary syngeneic murine breast tumors after daily i.v. dosing. Chol-DsiRNA Polyplexes, however, maintain LUC mRNA suppression ~48 h longer after the final dose than Chol-siRNA Polyplexes, suggesting they are a better candidate formulation. Here, we directly compared the activities of Chol-siRNA and Chol-DsiRNA Polyplexes in primary murine 4T1 breast tumors against STAT3, a therapeutically relevant target gene overexpressed in many solid tumors including breast cancer. We found that Chol-siSTAT3 Polyplexes suppressed STAT3 mRNA in 4T1 tumors with similar potency (half-maximal ED₅₀ 0.3 mg/kg) and kinetics over 96 hours as Chol-DsiSTAT3 Polyplexes but with slightly lower activity against total Stat3 protein (29% vs. 42% suppression) and tumor growth (11.5% vs. 8.6% rate-based T/C ratio) after repeated i.v. administration of tumor-saturating doses every other day. Thus, both Chol-siRNA Polyplexes and Chol-DsiRNA Polyplexes may be suitable clinical candidates for RNAi therapy of breast cancer and other solid tumors.

Generating long-lived mucosal and systemic antibodies through respiratory immunization with protective antigens encapsulated in nanoscale biodegradable particles could potentially decrease or eliminate the incidence of many infectious diseases but requires incorporation of a suitable mucosal immunostimulant. We previously found that respiratory immunization with a model protein antigen (LPS-free OVA) encapsulated in PLGA 50:50 nanoparticles (~380 nm diameter) surface modified with complement peptide-derived immunostimulant 02 (CPDI-02; formerly EP67) through 2kDa PEG linkers increases mucosal and systemic OVA-specific memory T-cells with long-lived surface phenotypes in young, naïve female C57BL/6 mice. Here, we determined if respiratory immunization with LPS-free OVA encapsulated in similar PLGA 50:50 microparticles (~1 μm diameter) surface modified with CPDI-02 (CPDI-02-MP) increases long-term OVA-specific mucosal and systemic antibodies. We found that, compared to MP surface modified with inactive, scrambled scCPDI-02 (scCPDI-02-MP), intranasal administration of CPDI-02-MP in 50 μL sterile PBS greatly increased titers of short-term (14 days post-immunization) and long-term (90 days post-immunization) antibodies against encapsulated LPS-free OVA in nasal lavage fluids, bronchoalveolar lavage fluids, and sera of young, naïve female C57BL/6 mice. Thus, surface modification of biodegradable microparticles with CPDI-02 is likely to increase long-term mucosal and systemic antibodies against encapsulated protein antigen after respiratory and possibly other routes of mucosal immunization.

Background/Objectives: Community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) greatly complicates the treatment of skin and soft tissue infections (SSTI). It was previously found that subcutaneous (SQ) treatment with the mononuclear phagocyte (MP)-selective activator complement peptide-derived immunostimulant-02 (CPDI-02; formerly EP67) increases prophylaxis of outbred CD-1 mice against SQ infection with CA-MRSA. Here, we determined if treatment with CPDI-02 also increases curative protection. Methods: Female CD-1 mice were challenged SQ with CA-MRSA USA300 LAC, then CPDI-02 or inactive scCPDI-02 was administered by a topical, SQ, IM, or IV route at 6 or 24 h post-challenge. Abscess sizes were compared over 10 days and CA-MRSA burden, neutrophils, MP, and pro-inflammatory cytokines were compared in subcutaneous abscesses. CPDI-02 PK and distribution in female CD-1 mice were compared after IM or IV dosing and CPDI-02 toxicity in male and female CD-1 mice was determined by IM dose escalation and repeat IM dosing. Results: Repeat IM treatment starting at 6 h post-challenge decreased maximum abscess surface area, CA-MRSA burden, and time to resolution, whereas repeat treatment by a topical, SQ, or IV route had no effect. Repeat treatment starting at 24 h post-challenge was ineffective by the current routes. Single IM treatment starting at 6 h post-challenge was as effective as repeat IM treatment, increased systemic exposure to CPDI-02, and, in subcutaneous abscesses, initially decreased IL-1β and increased MP. CPDI-02 was tolerated between 130 and 170 mg/kg after IM dose escalation and between 65 and 130 mg/kg after repeat IM dosing with males being more tolerant. Conclusions: Single early-stage IM treatment with CPDI-02 may increase curative protection against SSTI caused by CA-MRSA and/or other pathogens controlled by activated MP.

Community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) greatly complicates treatment of skin and soft tissue infections (SSTI). It was previously found that subcutaneous (SQ) treatment with the mononuclear phagocyte (MP)-selective activator, complement peptide-derived immunostimulant-02 (CPDI-02; formerly EP67), increases prophylaxis of outbred CD-1 mice against SQ infection with CA-MRSA. Here, we determined if treatment with CPDI-02 also increases curative protection. Methods: Female CD-1 mice were challenged SQ with CA-MRSA USA300 LAC then CPDI-02 or inactive scCPDI-02 was administered by a topical, SQ, IM, or IV route at 6- or 24-hrs post-challenge. Dermal abscess sizes were compared over 10 days and CA-MRSA burden, neutrophils, MP, and pro-inflammatory cytokines were compared in dermal abscesses. CPDI-02 PK and distribution in female CD-1 mice were compared after IM or IV dosing and CPDI-02 toxicity in male and female CD-1 mice was determined by IM dose escalation and repeat IM dosing. Results: Repeat IM treatment starting at 6-hrs post-challenge decreased maximum abscess surface area, CA-MRSA burden, and time to resolution, whereas repeat treatment by a topical, SQ, or IV route had no effect. Repeat treatment starting at 24-hrs post-challenge was ineffective by the current routes. Single IM treatment starting at 6-hrs post-challenge was as effective as repeat IM treatment, increased systemic exposure to CPDI-02, and, in dermal abscesses, initially decreased IL-1β and increased MP. CPDI-02 was tolerated to between 130-170 mg/kg after IM dose escalation and between 65-130 mg/kg after repeat IM dosing with males being more tolerant. Conclusions: Single early-stage IM treatment with CPDI-02 may increase curative protection against SSTI caused by CA-MRSA and/or other pathogens controlled by activated MP.