| Background and Objective:Chemotherapy remains as a major treatment modality for cancer treatment, yet its efficiency is limited by the associated toxic side effects as well as the evolution of the drug-resistant tumors after prolonged treatments. Also, chemotherapy is ineffective against certain tumor classes such as hepatocellular carcinoma, due to chemotherapy-deactivating enzymes liver cells.PDT has been attempted in clinical practice to combat cancers and showed limited efficacy in reversing drug resistance. As practiced in oncology a photosensitizing agent is applied and then activated by a specific wavelength and energy of light. This light energy in the presence of oxygen will lead to the creation of the photodynamic reaction, which is cyto and vasculo toxic. However, PDT remains the adjuvant therapy in clinically due to the poor solution ability and instability of the photosensitizer.The synergistic therapy, the combination of photo dynamic therapy and chemotherapy, using targeting drug delivery system (DDSs), has become a potential treatment in the battles with cancer. Recently, numerous nanoparticle-based DDSs have been developed to extend the blood circulation half-time, lower toxicity and increase tumor targeting ability. Nevertheless, few have resulted in significant improvements in patient survival rates. In addition, the biosafety of these nanoparticles remains to be addressed prior to their possible clinical applications.In this study, a natural molecule, hematoporphyrin (HP) based nanoparticle system was designed and synthesized to load doxorubicin (HPPD), in achieving a synergistic effect of chemotherapy and PDT. And its function in anti-tumor effect and drug resistance reversal will be studied in vitro and in vivo.Method:1) Poly ethylene glycol was employed to modify HP in order to form HPP nanoparticle. Nuclear magnetic resonance (NMR), transmission electronic microscopy (TEM) and dynamic light scattering (DLS) were used to analysis the structure, morphology and diameter of HPP nanoparticle, respectively. We also use High Performance Liquid Chromatography (HPLC) to investigate the drug release behavior of Dox loaded HPP.2) ADR/MCF-7cells were treated with various concentration of HPPD or free Dox, cell viability and50%inhibition concentration was calculated by MTT assay. Cellular uptake and intracellular location of HPPD was detected by flow cytometry analysis and confocal microscopy.3) Apoptotic cell percentage of evaluated by Anexin V/PI staining. We also study the subcellular distribution of cytochrome c after cell transfected with HPPDR. Key components of mitochondrial apoptosis pathway protein expression were detected by Western blotting.4) Subcutaneous ADR/MCF-7and MHCC-97H xenograft mouse model was used to evaluate the therapeutic efficacy of different Dox formulations. At day7after the last dosage, blood samples were obtained from all the mice for the measurement of blood cell counts, such as serum chemistry including creatine kinase (CK) and lactate dehydrogenase (LDH). Heart, liver, spleen, lung, kidney and tumor were collected, and all the tissues were homogenized. Dox was extracted from all tissues and detected with HPLC method to evaluate the drug distribution.5) Distribution of HPPD in tumor bearing mice was examined by tail-vein administrating of Cy5.5labeled HPPD, PEG or Cy5.5alone, respectively. And tumor-targeting ability of HPPD was evaluated by live animal imaging analysis.Results:1) PEG was successfully conjugated to HP, as evidenced by the13C-NMR spectrum. Analysis by DLS revealed that HPP formed narrowly dispersed nanoparticles with an average size of24.38±1.02nm. The result of TEM demonstrated that HPP formed small, spherical, narrowly dispersed nanoparticles, which possess a "core-shell" structure. Drug release rate of HPPD would be higher at the tumor sites than in circulation and further enhancement was detected when in combination with radiation.2) Encapsulation with HPP appeared to enhance the drug efficacy to some extent. Laser radiation further reduced the survival rate of cancer cells, resulting in a total of12-fold decrease in IC50values. Dox and HPPD (at the drug concentration of5μg/mL) resulted in cell death initially by15～18%and30～35%respectively (Figure2b). Laser radiation at100mW/cm2further enhanced the killing rate up to78%.3) The entry to nucleus of free Dox was inhibited in ADR/MCF-7cells. HPPD facilitated the enrichment of Dox inside the nucleus, and laser radiation treatment further increased nucleus distribution of Dox in ADR/MCF-7cells. Furthermore, flow cytometry analysis showed that the highest level of Dox fluorescence was detected in cells treated with HPPDR, further validating that the combined therapy had improved the retention of drugs inside the cells.4) Significant anti-tumor effect was observed in HPPDR-treated cells, with approximately42%cells apoptotic. Remarkable, release of the cytochrome c, a hallmark of (mitochondrial) apoptotic pathway, from mitochondria into the cytosol was observed by confocal fluorescent microscopy. Western blotting analysis further confirmed the enrichment of cytochrome c and apoptosis inducing factor (AIF) in cytosol.5) Free Dox or HPP with radiation failed to constrain the tumor growth. In sharp contrast, treatment with HPPD alone reduced the tumor size to an undetectable level during the period of3weeks. Addition of laser radiation further reduced this time period to2weeks, suggesting that HPPD-based PDT was effective in treating drug resistant tumors. HPPD also effectively inhibited tumor growth in MHCC-97H tumor planted mice.6) Live animal imaging analysis revealed that, in the control group, Cy5.5or PEGylated Cy5.5didn’t exhibit tumor targeting property24h after administration. Fluorescent signals were detected in tumors, kidneys, and spleen, but not in heart, liver, or lung in the group treated with HPPD-Cy5.52h after injection. In24h, the fluorescent signals in spleen and kidney were reduced while the signal in hepatoma maintained. Clearly, HPPD specifically targets hepatoma but not mouse liver. Morever, the similar distribution pattern was found in ADR/MCF-7tumor-bearing mice treated with HPPD. 7)24h after drug administration, tumors in HPPD-treated mice displayed a drug concentration at approximately32%of injected dose per gram of the tissues, which was2-3folds higher than that observed in the free Dox-treated mice, further validating the drug targeting property of the HPPD nanoparticles. Most importantly, ultra high tumor uptake was observed in diethylnitrosamine (DENA) induced orthotopic hepatocellular carcinoma marmoset model. And no fluorescence was found in normal liver.8) In both breast cancer and hepatoma tumor models, HPPDR yielded a remarkably lower level of CK/LDH, suggesting that HPPD based therapy significantly attenuated the toxic side effects. Hematoxylin and Eosin (H&E) staining revealed no detectable pathological effects or myocardial injury in HPPDR group. Meanwhile, remarkable nucleus atypia, increased necrotic cells and lower chromatin staining were observed in HPPDR-treated cells.Conclusions:To our knowledge, this is the first report of using hematoporphyrin, a safe and natural component in erythrocytes, as a drug delivery system. Dox loaded HPP can be easily synthesized and readily adopted for large-scale manufacture, acquiring the combinatorial efficacy of chemo-and photodynamic treatment, appears to be a promising strategy in combating drug-resistance tumors. |
PDF Full Text Request