Fabrication of Doxorubicin-Loaded Lipid-Based Nanocarriers by Microfluidic Rapid Mixing.

Doxorubicin (Dox) is a widely known chemotherapeutic drug that has been encapsulated into liposomes for clinical use, such as Doxil® and Myocet®. Both of these are prepared via remote loading methods, which require multistep procedures. Additionally, their antitumor efficacy is hindered due to the poor drug release from PEGylated liposomes in the tumor microenvironment. In this study, we aimed to develop doxorubicin-loaded lipid-based nanocarriers (LNC-Dox) based on electrostatic interaction using microfluidic technology. The resulting LNC-Dox showed high loading capacity, with a drug-to-lipid ratio (D/L ratio) greater than 0.2, and high efficacy of drug release in an acidic environment. Different lipid compositions were selected based on critical packing parameters and further studied to outline their effects on the physicochemical characteristics of LNC-Dox. Design of experiments was implemented for formulation optimization. The optimized LNC-Dox showed preferred release in acidic environments and better therapeutic efficacy compared to PEGylated liposomal Dox in vivo. Thus, this study provides a feasible approach to efficiently encapsulate doxorubicin into lipid-based nanocarriers fabricated by microfluidic rapid mixing.

A Novel Treatment Modality for Malignant Peripheral Nerve Sheath Tumor Using a Dual-Effect Liposome to Combine Photodynamic Therapy and Chemotherapy.

Neurofibromatosis type 1 (NF1) is an inherited neurological disorder. Approximately 5-13% of NF1 patients may develop a malignant peripheral nerve sheath tumor (MPNST), which is a neurofibrosarcoma transformed from the plexiform neurofibroma or schwannoma. Given the large size and easy metastasis of MPNST, it remains difficult to be cured by either surgical or conventional chemotherapy. In this study, we investigated the possibility of combining photodynamic therapy (PDT) and chemotherapy to treat MPNST by using a dual-effect liposome (named as PL-cDDP-Ce6), in which a chemotherapeutic agent, cisplatin (cDDP), and photosensitizer, chlorine e6 (Ce6) were encapsulated in the same carrier. The cytotoxic effect of PL-cDDP-Ce6 against MPNST cells was significantly higher than cells treated with liposomal cDDP or Ce6 alone or in combination after light irradiation. Treatment with the dual-effect liposomes in mice bearing xenograft MPNST tumor reveals a significant increase in survival rate compared to those treated with liposomal cDDP and Ce6 in combination. Moreover, there is no weight loss or derangements of serum biochemistry. In conclusion, this study demonstrates the clinical potential and advantage of using this liposomal drug for the treatment of MPNST.

Co-Encapsulation of Chlorin e6 and Chemotherapeutic Drugs in a PEGylated Liposome Enhance the Efficacy of Tumor Treatment: Pharmacokinetics and Therapeutic Efficacy.

Long-circulating PEG-modified liposome has been shown to improve pharmacokinetic properties and reduce systemic toxicity in cancer treatment. However, drug bioavailability from liposome remains a major challenge to the improvement of its therapeutic efficacy. Previously, we designed a PEGylated dual-effect liposome (named as PL-Dox-Ce6) with chlorin e6 incorporated in the lipid bilayer and Doxorubicin encapsulated in the interior. In this study, another dual-effect liposome with cisplatin encapsulated in the interior was further developed. The pharmacokinetics of these two dual-effect liposomes were studied in tumor-bearing mice. Based on the kinetic data of tumor and plasma, light irradiation was applied onto the tumors at different time points after drug administration to compare the therapeutic efficacy. We demonstrated that a single dose of the dual-effect liposomes combined with two doses of light irradiation can completely eradicate over 90% of the tumor in mice alone with significant survival rate and no toxicity. Thus, this study established a platform that utilizes the dual-effect liposome which combines photodynamic therapy and chemotherapy to improve the therapeutic outcomes of tumors.

Assessment of Photodynamic Inactivation against Periodontal Bacteria Mediated by a Chitosan Hydrogel in a 3D Gingival Model.

Chitosan hydrogels containing hydroxypropyl methylcellulose (HPMC) and toluidine blue O were prepared and assessed for their mucoadhesive property and antimicrobial efficacy of photodynamic inactivation (PDI). Increased HPMC content in the hydrogels resulted in increased mucoadhesiveness. Furthermore, we developed a simple In Vitro 3D gingival model resembling the oral periodontal pocket to culture the biofilms of Staphylococcus aureus (S. aureus), Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans), and Porphyromonas gingivalis (P. gingivalis). The PDI efficacy of chitosan hydrogel was examined against periodontal biofilms cultured in this 3D gingival model. We found that the PDI effectiveness was limited due to leaving some of the innermost bacteria alive at the non-illuminated site. Using this 3D gingival model, we further optimized PDI procedures with various adjustments of light energy and irradiation sites. The PDI efficacy of the chitosan hydrogel against periodontal biofilms can significantly improve via four sides of irradiation. In conclusion, this study not only showed the clinical applicability of this chitosan hydrogel but also the importance of the light irradiation pattern in performing PDI for periodontal disease.

Histone acetyltransferase p300 is induced by p38MAPK after photodynamic therapy: the therapeutic response is increased by the p300HAT inhibitor anacardic acid.

Oxidative stress mediated by photodynamic therapy (PDT) mediates the tumoricidal effect, but has also been shown to induce the expression of prosurvival molecules, such as cyclooxygenase-2 (COX-2), which is involved in tumor recurrences after PDT. However, the molecular mechanism is still not fully understood. In this study, we found that activated p38MAPK could significantly up-regulate the activity and expression of histone acetyltransferase p300 (p300HAT) in A375 and C26 cells treated with ALA-and chlorin e6 (Ce6)-mediated photodynamic treatment. A colony-formation assay showed that PDT-induced cytotoxicity was dramatically elevated in the presence of the p300HAT inhibitor anacardic acid (AA). Further studies showed that increased p300HAT acetylates histone H3 and NF-κB p65 subunit to up-regulate the COX-2 expression, which was reduced by AA or p300HAT shRNA. Using chromatin immunoprecipitation analysis, we found that the augmented acetylation of histone H3 and NF-κB increases their binding to the COX-2 promoter region. These in vitro findings were further verified in mice bearing murine C26 and human A375 tumors treated with liposomal Ce6 mediated PDT. Meanwhile, the combination of PDT and AA resulted in greater tumor regression in BALB/c mice bearing C26 tumors, compared with PDT only or combined with COX-2 inhibitor. Finally, we demonstrated that suppression of the PDT-induced p300HAT activity also resulted in the decreased expression of survivin, restoring caspase-3 activity and sensitizing PDT-treated cells from autophagy to apoptosis due to the Becline-1 cleavage. This study demonstrates for the first time the molecular mechanisms involved in histone modification induced by PDT-mediated oxidative stress, suggesting that HAT inhibitors may provide a novel therapeutic approach for improving PDT response.

Dual-effect liposomes encapsulated with doxorubicin and chlorin e6 augment the therapeutic effect of tumor treatment.

BACKGROUND AND OBJECTIVE: Long circulating doxorubicin (Dox)-loaded PEGylated liposomes are clinically safer than the free form due to the significant reduction of cardiac toxicity. However, the therapeutic efficacy of the PEGylated liposome could further be improved if poor diffusivity and slow drug release of the liposome in tumor interstitium can be overcome. In this study, a dual-effect liposome triggered by photodynamic effect was developed to improve the therapeutic efficacy of Dox-loaded PEGylated liposomes. MATERIALS AND METHODS: Dox and chlorin e6 (Ce6) were co-encapsulated in PEGylated liposomes (named as PL-Dox-Ce6). To induce the drug release, photodynamic effect was triggered by the light irradiation of a 662 nm diode laser. The cellular distribution of Dox and Ce6 was examined under confocal microscope. The in vitro and in vivo cytotoxicity of PL-Dox-Ce6 was determined via the colony formation assay and the synergistic C26 tumor model, respectively. RESULTS: The cellular distribution of PL-Dox-Ce6 was in the cytoplasmic area; while under light irradiation, Dox was co-localized with nuclear staining positive signals. The cellular cytotoxicity of PL-Dox-Ce6 was significantly higher than the controls including liposomes encapsulating either Dox (PL-Dox) or Ce6 (PL-Ce6). The in vivo treatment efficacy of PL-Dox-Ce6 determined in the C26 tumor model reveals a significant therapeutic effect compared to that of PL-Ce6 and PL-Dox alone or in combination. CONCLUSION: This study indicates that this dual-effect PEGylated liposome could provide clinical advantages in the combination regimen of photodynamic therapy and chemotherapy.

Soluble AXL: a possible circulating biomarker for neurofibromatosis type 1 related tumor burden.

Neurofibromatosis type 1 (NF1) is the most common tumor predisposition disorder affecting 1/3500 worldwide. Patients are at risk of developing benign (neurofibromas) and malignant peripheral nerve sheath tumors (MPNST). The AXL receptor tyrosine kinase has been implicated in several kinds of cancers, but so far no studies have investigated the role of AXL in NF1 related tumorigenesis. Recently, the soluble fraction from the extracellular domain of AXL (sAXL) has been found in human plasma, and its level was correlated to poor prognosis in patients with renal cancer. Compared to normal human Schwann cells, a significantly high expression level of AXL was found in three of the four MPNST cell lines and two of the three primary MPNST tissues. Similarly, the level of sAXL in conditioned media corresponded to the protein and mRNA levels of AXL in the MPNST cell lines. Furthermore, in two different human MPNST xenograft models, the human sAXL could be detected in the mouse plasma. Its level was proportionate to the size of the xenograft tumors, while no human sAXL was detect prior to the formation of the tumors. Treatment with a newly developed photodynamic therapy, prevented further tumor growth and resulted in drastically reduced the levels of sAXL compared to that of the control group. Finally, the level of sAXL was significantly increased in patients with plexiform tumors compared to patients with only dermal neurofibromas, further supporting the role of sAXL as a marker for NF1 related tumor burden.