Liposomes Enhance Dye Localization within the Mammary Ducts of Porcine Nipples.

Transductal and transepidermal diffusion are two distinct penetration routes of molecules administered via the nipple. To improve the therapeutic potential of this drug administration technique, drug penetration into the mammary ducts should be maximized, which may be accomplished through design optimization of drug delivery vehicles. In this study, we evaluated liposomes, ranging in size from 100 to 3000 nm, to improve ductal penetration of model fluorescent dyes using fluorescence microscopy and image analysis. Liposomes encapsulating a model fluorescent lipophilic dye, nile red, or hydrophilic dye, sulforhodamine B, were applied topically on porcine nipples for 6 h in vitro. Liposome encapsulation of sulforhodamine B significantly reduced the total amount of dye penetrating the nipple, while penetration of liposome-encapsulated nile red varied depending on vesicle size, as compared to their solution controls. However, the fluorescence intensity localized at the ductal epithelium was higher at extended nipple depths in tissues treated with liposomes versus dye solutions, suggesting a higher concentration of dye penetrating the nipple via the ducts. In contrast, the fluorescence intensity measured at the stratum corneum was reduced (sulforhodamine B) or unchanged (nile red) in nipples treated with liposomes versus dye solutions, suggesting a decrease or no change in dye penetration of the nipple via the stratum corneum. Furthermore, the limited penetration distance into the connective tissue beyond the ductal epithelium for both liposome-encapsulated nile red and sulforhodamine B suggests that liposomes remain intact over the 6 h duration of this study when penetrating through the ducts and enhance retention within the ductal lumen. However, the varied penetration profiles into the connective tissue beyond the stratum corneum between liposome-encapsulated nile red and sulforhodamine B suggests that the liposomes destabilize when penetrating the outer tissues layers of the nipple. Overall, liposomes, regardless of size, improved penetration into and retention within the mammary ducts, while limiting penetration into the stratum corneum, indicating their capacity to target the mammary ductal network.

Tumor-derived granulocyte colony-stimulating factor diminishes efficacy of breast tumor cell vaccines.

BACKGROUND: Although metastasis is ultimately responsible for about 90% of breast cancer mortality, the vast majority of breast-cancer-related deaths are due to progressive recurrences from non-metastatic disease. Current adjuvant therapies are unable to prevent progressive recurrences for a significant fraction of patients with breast cancer. Autologous tumor cell vaccines (ATCVs) are a safe and potentially useful strategy to prevent breast cancer recurrence, in a personalized and patient-specific manner, following standard-of-care tumor resection. Given the high intra-patient and inter-patient heterogeneity in breast cancer, it is important to understand which factors influence the immunogenicity of breast tumor cells in order to maximize ATCV effectiveness. METHODS: The relative immunogenicity of two murine breast carcinomas, 4T1 and EMT6, were compared in a prophylactic vaccination-tumor challenge model. Differences in cell surface expression of antigen-presentation-related and costimulatory molecules were compared along with immunosuppressive cytokine production. CRISPR/Cas9 technology was used to modulate tumor-derived cytokine secretion. The impacts of cytokine deletion on splenomegaly, myeloid-derived suppressor cell (MDSC) accumulation and ATCV immunogenicity were assessed. RESULTS: Mice vaccinated with an EMT6 vaccine exhibited significantly greater protective immunity than mice vaccinated with a 4T1 vaccine. Hybrid vaccination studies revealed that the 4T1 vaccination induced both local and systemic immune impairments. Although there were significant differences between EMT6 and 4T1 in the expression of costimulatory molecules, major disparities in the secretion of immunosuppressive cytokines likely accounts for differences in immunogenicity between the cell lines. Ablation of one cytokine in particular, granulocyte-colony stimulating factor (G-CSF), reversed MDSC accumulation and splenomegaly in the 4T1 model. Furthermore, G-CSF inhibition enhanced the immunogenicity of a 4T1-based vaccine to the extent that all vaccinated mice developed complete protective immunity. CONCLUSIONS: Breast cancer cells that express high levels of G-CSF have the potential to diminish or abrogate the efficacy of breast cancer ATCVs. Fortunately, this study demonstrates that genetic ablation of immunosuppressive cytokines, such as G-CSF, can enhance the immunogenicity of breast cancer cell-based vaccines. Strategies that combine inhibition of immunosuppressive factors with immune stimulatory co-formulations already under development may help ATCVs reach their full potential.

Evaluation of Amphiphilic Star/Linear-Dendritic Polymer Reverse Micelles for Transdermal Drug Delivery: Directing Carrier Properties by Tailoring Core versus Peripheral Branching.

The reverse micelle self-assembly of lipophile-functionalized poly(ethylene glycol) (PEG) dendrimer hybrids is probed for applications in carrier-mediated transdermal drug delivery. Under investigation are topologically diverse amphiphiles featuring controlled branching motifs at either the polymer core (one-, two-, and four-arm PEG) and the polar/nonpolar interface (peripheral dendritic generations 0-2). Thus, a systematic investigation of the effect of branching location (core vs peripheral) on carrier properties is described. Dye-encapsulation experiments verify these materials are capable of forming well-defined aggregates and solubilizing polar compounds. Further quantification of reverse micelle critical micelle concentration and dye loading capacity for the branched amphiphile library was obtained through spectroscopy characterization. Both core and peripheral branching are shown to significantly influence dynamic encapsulation behavior, with evidence of location-based contributions extending beyond multiplicity of branching alone. Finally, the in vitro transdermal diffusion of the reverse micelle carriers was investigated through Franz diffusion cell experiments using physiologically relevant juvenile porcine dermis. The permeation results, combined with previously reported aggregate size trends, show the complex relationship between polymer branching and transdermal transport, with the lowest core- and highest peripherally-branched amphiphilic analogs exhibiting optimal transdermal permeation characteristics for this set of branched carriers.

Intravesical chitosan/interleukin-12 immunotherapy induces tumor-specific systemic immunity against murine bladder cancer.

Bladder cancer is a highly recurrent disease in need of novel, durable treatment strategies. This study assessed the ability of an intravesical immunotherapy composed of a coformulation of the biopolymer chitosan with interleukin-12 (CS/IL-12) to induce systemic adaptive tumor-specific immunity. Intravesical CS/IL-12 immunotherapy was used to treat established orthotopic MB49 and MBT-2 bladder tumors. All mice receiving intravesical CS/IL-12 immunotherapy experienced high cure rates of orthotopic disease. To investigate the durability and extent of the resultant adaptive immune response, cured mice were rechallenged both locally (intravesically) and distally. Cured mice rejected 100 % of intravesical tumor rechallenges and 50-100 % of distant subcutaneous rechallenges in a tumor-specific manner. The ability of splenocytes from cured mice to lyse targets in a tumor-specific manner was assessed in vitro, revealing that lytic activity of splenocytes from cured mice was robust and tumor specific. Protective immunity was durable, lasting for at least 18 months after immunotherapy. In an advanced bladder cancer model, intravesical CS/IL-12 immunotherapy controlled simultaneous orthotopic and subcutaneous tumors in 70 % of treated mice. Intravesical CS/IL-12 immunotherapy creates a robust and durable tumor-specific adaptive immune response against bladder cancer. The specificity, durability, and potential of this therapy to treat both superficial and advanced disease are deserving of consideration for clinical translation.

Nanoemulsions Enhance in vitro Transpapillary Diffusion of Model Fluorescent Dye Nile Red.

While the feasibility of transpapillary drug delivery has previously been established, localized transport via the mammary ducts may be improved with tailored drug delivery formulations. The objective of this study was to investigate the impact of nanoemulsion encapsulation on transpapillary delivery in vitro. Nanoemulsion formulations composed of isopropyl myristate and Tween 80 encapsulating a fluorescent dye were applied topically on porcine nipples using a Franz diffusion cell. A combination of dye extraction and fluorescence image analysis was used to quantify the total amount of dye retained within the nipple and to characterize the penetration routes. After diffusion for 6 hours, the amount of dye deposited in the nipple was proportional to the formulation's water concentration. The 90% water formulation deposited significantly more dye via both the stratum corneum and mammary ducts, while the 80% and 70% water formulations moderately increased ductal penetration, but minimally altered stratum corneum penetration as compared to the control solution. Similar trends were found after diffusion for 48 hours; however, the overall impact was diminished, likely due to the nanoemulsion's topical instability. This study indicates that drug delivery vehicles, nanoemulsions specifically, enhance delivery of encapsulated molecules via the stratum corneum and mammary ducts in a formulation-dependent basis.

Determination of permeation pathways of hydrophilic or hydrophobic dyes through the mammary papilla.

The transport pathways and permeation kinetics of lipophilic and hydrophilic fluorescent dyes through porcine mammary papillae were visualized and quantified. Porcine mammary papillae, removed from full-thickness abdominal tissue, were positioned in a Franz diffusion cell for passive diffusion studies. Solutions containing the fluorescent dyes were applied topically for time periods ranging from 30 min to 48 h. Dye concentrations in tissue and Franz diffusion compartments were analyzed using fluorescence microscopy and fluorimetry. Fluorescence micrographs elucidated two permeation pathways, transepidermal and transductal. Hydrophilic sulforhodamine B predominantly penetrated via the transepidermal route, while lipophilic nile red diffused mainly by the transductal route. An almost 4-fold higher amount of sulforhodamine B was retained within the nipple over time compared to nile red, despite both dyes permeating through the tissue at similar rates. Diffusion through the porcine nipple was 500-fold higher than through adjacent skin for both dyes, likely attributable to the two mammary ducts which provide an entry point and transport route through the tissue. These results, generated from both qualitative and quantitative evidence at a micro and macro scale, demonstrate that the mammary ducts provide a direct pathway that contributes significantly to passive transport through the nipple, particularly for lipophilic molecules.

Current status of autologous breast tumor cell-based vaccines.

Approximately nine out of ten breast cancer-related deaths are attributable to metastasis. Yet, less than 4% of breast cancer patients are initially diagnosed with metastatic cancer. Therefore, the majority of breast cancer-related deaths are due to recurrence and progression of non-metastatic disease. There is tremendous clinical opportunity for novel adjuvant strategies, such as immunotherapies, that have the potential to prevent progressive recurrences. In particular, autologous tumor cell-based vaccines (ATCVs) can train a patient's immune system to recognize and eliminate occult disease. ATCVs have several advantages including safety, multivalency and patient specificity. Furthermore, because lumpectomy or mastectomy is indicated for the vast majority of breast cancer patients, resected tumors offer a readily available, patient-specific source of tumor antigen. Disadvantages of ATCVs include poor immunogenicity and production inconsistencies. This review summarizes recent progress in the development of autologous breast tumor vaccines and offers insight for overcoming existing limitations.

Neoadjuvant immunotherapy with chitosan and interleukin-12 to control breast cancer metastasis.

Metastasis accounts for approximately 90% of breast cancer-related deaths. Therefore, novel approaches which prevent or control breast cancer metastases are of significant clinical interest. Interleukin-12 (IL-12)-based immunotherapies have shown promise in controlling metastatic disease, yet modest responses and severe toxicities due to systemic administration of IL-12 in early trials have hindered clinical application. We hypothesized that localized delivery of IL-12 co-formulated with chitosan (chitosan/IL-12) could elicit tumor-specific immunity and provide systemic protection against metastatic breast cancer while minimizing systemic toxicity. Chitosan is a biocompatible polysaccharide derived primarily from the exoskeletons of crustaceans. In a clinically relevant resection model, mice bearing spontaneously metastatic 4T1 mammary adenocarcinomas received intratumoral injections of chitosan/IL-12, or appropriate controls, prior to tumor resection. Neoadjuvant chitosan/IL-12 immunotherapy resulted in long-term tumor-free survival in 67% of mice compared to only 24% or 0% of mice treated with IL-12 alone or chitosan alone, respectively. Antitumor responses following chitosan/IL-12 treatment were durable and provided complete protection against rechallenge with 4T1, but not RENCA renal adenocarcinoma, cells. Lymphocytes from chitosan/IL-12-treated mice demonstrated robust tumor-specific lytic activity and interferon-γ production. Cell-mediated immune memory was confirmed in vivo via clinically relevant delayed-type hypersensitivity (DTH) assays. Comprehensive hematology and toxicology analyses revealed that chitosan/IL-12 induced transient, reversible leukopenia with no changes in critical organ function. Results of this study suggest that neoadjuvant chitosan/IL-12 immunotherapy prior to breast tumor resection is a promising translatable strategy capable of safely inducing to tumor-specific immunity and, in the long term, reducing breast cancer mortality due to progressive recurrences.