Yüzeyi modifiye edilmiş antineoplastik ajan içeren katı lipid nanopartikül formulasyonların geliştirilmesi ve melanomda etkisinin değerlendirilmesi
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Dosyalar
Tarih
2020
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Yayıncı
Ege Üniversitesi, Sağlık Bilimleri Enstitüsü
Erişim Hakkı
info:eu-repo/semantics/embargoedAccess
Özet
Kanser, küresel olarak ölümlerin en önde gelen nedenlerinden biri olmakla birlikte; altı ölümden biri neoplazmla ilişkilidir. Günümüzde, her yıl 2 ile 3 milyon kişide me-lanom dışı cilt kanseri ve 132.000 kişide melanom cilt kanseri görülmekte; her üç kan-serden biri cilt kanseri olarak teşhis edilmektedir. Melanom, sağlıklı melanositlerin maligniteye dönüşümü ile oluşan bir kanser türüdür. Ciltte ve mukozada görülen me-lanomlar, lenfatik sistemle bölgesel lenf nodlarına metastaz yapma eğilimindedir. Bir-çok molekül, kanser tedavisi için geliştirilmeye çalışılmış olsa da, düşük çözünürlük ve yan etki göstermeleri nedeniyle, kanser tedavisine yönelik konvansiyonel uygula-malar yetersiz kalmaktadır. İlaçların vücutta spesifik olmayan dağılımları ve hedef bölgeye gidememesi, terapötik etkinliğin ve biyoyararlanımın azalmasına neden ol-makta; hedefli ilaç iletim sistemlerine ihtiyaç duyulmaktadır.
Günümüzde tedavi seçenekleri arasında, immünoterapi, hedeflenmiş tedaviler ve ke-moterapi yerini almaktadır. İmmünoterapötik olarak önemli bir grup olan Programlan-mış Ölüm Proteini 1 (Programmed Death 1 Protein (PD-1)), tümör hücrelerince eksp-rese edilen Programlanmış Ölüm Ligandı 1’e (PD-L1) bağlanarak T lenfositleri baskı-lamakta ve immünsupresyona yol açmaktadır. 4-1BB antikorları ise, T hücrelerini sti-müle ederek immün sistemi aktive etmektedir. Kemoterapötik bir ajan olarak all-trans retinoik asit (ATRA), retinoik asit grubundandır. Vesanoid adıyla onaylı bir ilaç olarak kanser kemoterapisinde kullanılmaktadır. Ayrıca, nanopartiküllerin formulasyonunda kullanılan sfingomiyelinler, hücre siklüsünü etkileyen tümör-baskılayıcı lipidlerdir.
Çalışmamızda, melanom için, antikor konjuge edilmiş, antineoplastik ajan yüklü katı lipid nanopartiküller (KLN) ile hem hedefe yönelik kemoterapötik hem de immünote-rapötik bir yaklaşımla etkin ve güvenilir bir tedavi geliştirilmesi amaçlanmıştır. Böy-lece bölgede monoklonal antikorlar aracılığı ile immümodulasyonun, ve ATRA yüklü KLN'leriyle antitümör etkinlik sağlanması hedeflenmiştir. KLN'lerin karakterizasyon çalışmaları ve melanom hücreleri üzerindeki antikanser etkileri, in vitro ve primer kültürden elde edilen ex vivo hücre kültürü çalışmaları ile desteklenmiştir. Deneysel çalışmamızda, yüzeyi modifiye edilmiş ATRA yüklü KLN'ler hazırlanmış ve KLN'lere anti-PD-L1 ve anti-4-1BB antikorları konjuge edilmiştir. KLN formülas-yonlarının karakterizasyon çalışması yapılmış ve stabiliteleri 9 ay boyunca incelen-miştir. Optimize edilen KLN formulasyonlarının B16F10 melanom hücrelerindeki et-kinliği, in vitro hücre kültürü çalışmaları ile belirlenmiştir. Verilere göre, ATRA yüklü KLN'lerin hücreler ile 1 saatlik inkübasyonu sonucunda, neredeyse tamamının hücre içine alındığı gösterilmiştir. Melanom hücrelerinin ATRA yüklü KLN ile 24 saatlik inkübasyonunda, hücrelerin G2/M fazında hücre döngüsü arestine uğradığı ve kontrol grubuna göre anlamlı bir farkla apoptoza gittiği görülmüştür. Bu veriler, qPCR verileri ile desteklenmiş, p53 ve Bax gen ekspresyonlarında kontrole göre anlamlı bir artış, Bcl-2 gen ekspresyonunda ise anlamlı bir azalma görülmüştür. Antikor bağlı ATRA yüklü KLN çalışmalarında, KLN'lerin melanom üzerindeki PD-L1'e bağlandığı göste-rilmiştir. Aynı zamanda, splenosit ve melanom hücreleri ile yapılan ko-kültür çalışma-larında, formülasyonların B16F10 melanom hücreleri üzerindeki sitotoksik etkileri konmuştur.
Çalışmanın sonucunda, melanom hücrelerindeki PD-L1'e hedefli, anti-PD-L1 ve anti-4-1BB monoklonal antikorları konjuge edilmiş ATRA yüklü KLN sistemleri gelişti-rilmiş, melanom tedavisinde kullanılmak üzere, hedefe yönelik potansiyel bir nano-partiküler sistemin etkinliği ortaya konmuştur. Melanom tedavisine immünoterapötik ve kemoterapötik bir yaklaşım içeren, umut vaadeden bir formulasyon olduğu sonu-cuna varılmıştır.
Cancer is one of the leading causes of deaths globally; one of six deaths is associated with neoplasm. Non-melanoma skin cancer is observed in 2-3 million people each year and melanoma skin cancer in 132,000 people; one of every three cancers is diagnosed in the skin. Melanoma occurs with malignant transformation of normal melanocytes. Skin and mucosal melanoma tends to metastasize to the regional lymph nodes with the lymphatic system in early stages of the progression. Although many agents have been tried to be developed for the treatment, conventional applications are insufficient due to the low solubility and side effects. Non-specific distribution of drugs in the body and inability to reach to the target area lead to decreased therapeutic efficacy and bio-availability; and thus, targeted delivery systems become necessary. Treatment strategies include immunotherapy, targeted therapy, and chemotherapy. Among these therapies, Programmed cell death protein 1 (PD-1) binds to programmed cell death-ligand 1 (PD-L1) expressed by tumor cells, suppressing T lymphocytes, le-ading to immunosuppression. 4-1BB antibodies activate the immune system by stimu-lating T cells. All-trans retinoic acid (ATRA) belongs to retinoic acid group and used in cancer chemotherapy with the approved medicine Vesanoid®. In addition, sphin-gomyelins used in nanoparticle formulations are tumor-suppressor lipids. In the study, it was aimed to develop an effective and reliable treatment for melanoma using antibody-conjugated, antineoplastic agent-loaded solid lipid nanoparticles (SLN) with a targeted chemotherapeutic and immunotherapeutic approach. Thus, both antitumor activity and immunomodulation was provided by monoclonal antibody con-jugated-ATRA-loaded SLN. Characterization studies of SLNs and their anticancer ef-fects on melanoma cells were supported by both in vitro and ex vivo primary cell cul-ture studies. In the experimental part, surface-modified ATRA-loaded SLNs were prepared, and anti-PD-L1 and anti-4-1BB antibodies were conjugated to the SLNs. SLN formulati-ons were characterized for the physicochemical properties and stability for 9 months. The effect of optimized SLN formulations on B16F10 melanoma cells was determined by in vitro cell culture studies. It has been shown that all ATRA-loaded SLN formula-tions were observed to be taken up by the cell within 1 hour-incubation. After 24-hour incubation, cells were induced to cell cycle arrest in G2/M phase that is followed by a significant apoptotic cell death when compared to control group. Cell cycle and apop-tosis data were supplemented with qPCR results indicating a significant increase in p53 and Bax gene expressions, and a significant decrease in Bcl-2 expression compa-red to control. Conjugation of anti-PD-L1 and anti-4-1BB antibodies to ATRA-loaded SLN studies resulted in binding of SLNs to PD-L1 expressed on the melanoma cell membrane, indicating that targeted therapy was successfully achieved. In parallel, co-culture studies including splenocytes and melanoma cells demonstrated the cytotoxic effect of the formulations on B16F10 melanoma cells. In conclusion, anti-PD-L1 and anti-4-1BB monoclonal antibodies-conjugated ATRA-loaded SLNs were developed successfully and specific targeting to PD-L1 on mela-noma cells was achieved. The efficiency on cell culture studies has further highlighted the promise of the developed nanoparticular formulation for the treatment of mela-noma. The developed methodology is particularly unique due to applying both immu-notherapeutic and chemotherapeutic approaches together for the treatment.
Cancer is one of the leading causes of deaths globally; one of six deaths is associated with neoplasm. Non-melanoma skin cancer is observed in 2-3 million people each year and melanoma skin cancer in 132,000 people; one of every three cancers is diagnosed in the skin. Melanoma occurs with malignant transformation of normal melanocytes. Skin and mucosal melanoma tends to metastasize to the regional lymph nodes with the lymphatic system in early stages of the progression. Although many agents have been tried to be developed for the treatment, conventional applications are insufficient due to the low solubility and side effects. Non-specific distribution of drugs in the body and inability to reach to the target area lead to decreased therapeutic efficacy and bio-availability; and thus, targeted delivery systems become necessary. Treatment strategies include immunotherapy, targeted therapy, and chemotherapy. Among these therapies, Programmed cell death protein 1 (PD-1) binds to programmed cell death-ligand 1 (PD-L1) expressed by tumor cells, suppressing T lymphocytes, le-ading to immunosuppression. 4-1BB antibodies activate the immune system by stimu-lating T cells. All-trans retinoic acid (ATRA) belongs to retinoic acid group and used in cancer chemotherapy with the approved medicine Vesanoid®. In addition, sphin-gomyelins used in nanoparticle formulations are tumor-suppressor lipids. In the study, it was aimed to develop an effective and reliable treatment for melanoma using antibody-conjugated, antineoplastic agent-loaded solid lipid nanoparticles (SLN) with a targeted chemotherapeutic and immunotherapeutic approach. Thus, both antitumor activity and immunomodulation was provided by monoclonal antibody con-jugated-ATRA-loaded SLN. Characterization studies of SLNs and their anticancer ef-fects on melanoma cells were supported by both in vitro and ex vivo primary cell cul-ture studies. In the experimental part, surface-modified ATRA-loaded SLNs were prepared, and anti-PD-L1 and anti-4-1BB antibodies were conjugated to the SLNs. SLN formulati-ons were characterized for the physicochemical properties and stability for 9 months. The effect of optimized SLN formulations on B16F10 melanoma cells was determined by in vitro cell culture studies. It has been shown that all ATRA-loaded SLN formula-tions were observed to be taken up by the cell within 1 hour-incubation. After 24-hour incubation, cells were induced to cell cycle arrest in G2/M phase that is followed by a significant apoptotic cell death when compared to control group. Cell cycle and apop-tosis data were supplemented with qPCR results indicating a significant increase in p53 and Bax gene expressions, and a significant decrease in Bcl-2 expression compa-red to control. Conjugation of anti-PD-L1 and anti-4-1BB antibodies to ATRA-loaded SLN studies resulted in binding of SLNs to PD-L1 expressed on the melanoma cell membrane, indicating that targeted therapy was successfully achieved. In parallel, co-culture studies including splenocytes and melanoma cells demonstrated the cytotoxic effect of the formulations on B16F10 melanoma cells. In conclusion, anti-PD-L1 and anti-4-1BB monoclonal antibodies-conjugated ATRA-loaded SLNs were developed successfully and specific targeting to PD-L1 on mela-noma cells was achieved. The efficiency on cell culture studies has further highlighted the promise of the developed nanoparticular formulation for the treatment of mela-noma. The developed methodology is particularly unique due to applying both immu-notherapeutic and chemotherapeutic approaches together for the treatment.
Açıklama
Anahtar Kelimeler
Melanom, Katı Lipid Nanopartikül, All-trans Retinoik Asit, Anti-PD-L1, Anti-4-1BB, Solid Lipid Nanoparticle
