Nghiên cứu quy trình chế tạo và đánh giá hiệu quả tác động của hệ nano đa chức năng (polymer drug fe3o4 folate) lên tế bào ung thư

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Nghiên cứu quy trình chế tạo và đánh giá hiệu quả tác động của hệ nano đa chức năng (polymer drug fe3o4 folate) lên tế bào ung thư

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BỘ GIÁO DỤC VÀ ĐÀO TẠO VIỆN HÀN LÂM KHOA HỌC VÀ CÔNG NGHỆ VIỆT NAM HỌC VIỆN KHOA HỌC VÀ CÔNG NGHỆ - LÊ THỊ THU HƢƠNG NGHIÊN CỨU CHẾ TẠO VÀ ĐÁNH GIÁ HIỆU QUẢ TÁC ĐỘNG CỦA HỆ NANO ĐA CHỨC NĂNG (POLYMER-DRUG- Fe3O4-FOLATE) LÊN TẾ BÀO UNG THƢ LUẬN ÁN TIẾN SỸ KHOA HỌC VẬT LIỆU HÀ NỘI – 2018 BỘ GIÁO DỤC VÀ ĐÀO TẠO VIỆN HÀN LÂM KHOA HỌC VÀ CÔNG NGHỆ VIỆT NA HỌC VIỆN KHOA HỌC VÀ CÔNG NGHỆ - LÊ THỊ THU HƢƠNG NGHIÊN CỨU CHẾ TẠO VÀ ĐÁNH GIÁ HIỆU QUẢ TÁC ĐỘNG CỦA HỆ NANO ĐA CHỨC NĂNG (POLYMER-DRUG- Fe3O4-FOLATE) LÊN TẾ BÀO UNG THƢ LUẬN ÁN TIẾN SỸ KHOA HỌC VẬT LIỆU Chuyên ngành: Vật liệu điện tử Mã số: 9440123 Ngƣời hƣớng dẫn khoa học: TS Hà Phƣơng Thƣ GS.TSKH Nguyễn Xuân Phúc Hà Nội – 2018 LỜI CAM ĐOAN Tôi xin cam Ďoan Ďây công trình nghiên cứu riêng tơi dƣới hƣớng dẫn TS Hà Phƣơng Thƣ GS.TSKH Nguyễn Xuân Phúc khơng trùng lặp với cơng trình khoa học khác Các số liệu kết nghiên cứu nêu luận án trung thực, Ďƣợc Ďồng tác giả cho phép sử dụng chƣa Ďƣợc sử dụng Ďể bảo vệ học vị nào, chƣa Ďƣợc cơng bố cơng trình khác Hà Nội, tháng năm 2018 Tác giả luận án Lê Thị Thu Hƣơng LỜI CẢM ƠN Tôi xin bày tỏ lòng biết ơn chân thành sâu sắc Ďến tập thể giáo viên hƣớng dẫn, TS Hà Phƣơng Thƣ GS TSKH Nguyễn Xuân Phúc Ďã tận tình hƣớng dẫn, hỗ trợ Ďịnh hƣớng cho suốt thời gian thực luận án Đặc biệt, xin chân thành cảm ơn hỗ trợ kinh phí từ Ďề tài KHCN mã số 106-YS.06-2015.14 (HPT) Ďề án 911 Xin chân thành cảm ơn Ban lãnh Ďạo Khoa Khoa học vật liệu lƣợng Học viện Khoa học Cơng nghệ Phòng Vật liệu Nano Y sinh, Phòng thí nghiệm trọng Ďiểm - Viện Khoa học Vật liệu, Viện Hàn làm Khoa học Công nghệ Việt Nam Ďã tạo Ďiều kiện thuận lợi sở vật chất cho tơi suốt q trình thí nghiệm Ďóng góp ý kiến chun mơn suốt q trình thực bảo vệ Luận án Tôi xin chân thành cảm ơn Ban Giám Ďốc, BCN Khoa Môi trƣờng tập thể Bộ môn Hố học, Học viện Nơng nghiệp Việt Nam, Ďã tạo Ďiều kiện giúp Ďỡ tơi q trình cơng tác Ďể tơi hồn thành luận án Xin cảm ơn Ban lãnh Ďạo phận Đào tạo Viện Khoa học Vật liệu Ďã hỗ trợ tơi hồn thành học phần luận án thủ tục cần thiết khác trình thực luận án Cuối xin bày tỏ lời cảm ơn sâu sắc Ďến gia Ďình, Ďã ln chia sẻ, Ďộng viên tinh thần nguồn cổ vũ, giúp Ďỡ vƣợt qua khó khăn suốt q trình thực Luận án Hà nội, ngày tháng năm 2018 Nghiên cứu sinh Lê Thị Thu Hƣơng i MỤC LỤC DANH MỤC CÁC KÍ HIỆU, CÁC CHỮ VIẾT TẮT .v DANH MỤC CÁC BẢNG viii DANH MỤC CÁC HÌNH VẼ, ĐỒ THỊ .x MỞ ĐẦU .1 CHƢƠNG 1: TỔNG QUAN .4 1.1 Khái quát hệ vật liệu nano y sinh 1.1.1 Cấu trúc hệ nano y sinh 1.1.2 Các chức y sinh hệ .6 1.2 Phƣơng pháp tổng hợp hạt nano Fe3O4 1.2.1 Phƣơng pháp Ďồng kết tủa 1.2.2 Phƣơng pháp thuỷ nhiệt 1.2.3 Phƣơng pháp phân huỷ nhiệt .9 1.2.4 Sử dụng kĩ thuật vi sóng tổng hợp Fe3O4 10 1.3 Tính chất ứng dụng hạt nano Fe3O4 y sinh học 11 1.3.1 Một số tính chất từ hạt nano oxit sắt từ Fe3O4 11 1.3.2 Mang thuốc hƣớng Ďích 13 1.3.3 Nhiệt trị phóng thích thuốc dựa hiệu ứng Ďốt nóng cảm ứng từ 15 1.3.4 Tăng cƣờng Ďộ tƣơng phản ảnh cộng hƣởng từ hạt nhân .18 1.3.5 Hệ nano Ďa chức .20 1.4 Vấn Ďề hạt nano oxit sắt từ cho ứng dụng y sinh 23 1.5 Chức hoá bề mặt hạt Fe3O4 .25 1.5.1 Bền hoá hạt nano Fe3O4 polime tổng hợp 25 1.5.2 Bền hoá hạt nano Fe3O4 polime tự nhiên .26 1.5.3 Kết hợp thuốc chống ung thƣ 29 1.5.4 Yếu tố hƣớng Ďích folate 31 Kết luận chƣơng .33 CHƢƠNG 2: VẬT LIỆU VÀ PHƢƠNG PHÁP NGHIÊN CỨU 34 2.1 Tổng hợp vật liệu .34 2.1.1 Nguyên vật liệu .34 2.1.2 Tổng hợp hạt nano oxit sắt từ phƣơng pháp Ďồng kết tủa 34 ii 2.1.3 Tổng hợp hạt nano Fe3O4 theo phƣơng pháp Ďồng kết tủa sử dụng kĩ thuật vi sóng 34 2.1.4 Bọc hạt nano Fe3O4 polime sinh học 36 2.1.5 Mang thuốc Curcumin Doxorubicin lên hệ 36 2.1.6 Gắn yếu tố hƣớng Ďích folate 37 2.1.7 Kết hợp chấm lƣợng tử CdTe 38 2.2 Các phƣơng pháp Ďặc trƣng tính chất hệ 39 2.2.1 Nhiễu xạ tia X 39 2.2.2 Phổ hấp thụ hồng ngoại 40 2.2.3 Phổ UV-Vis huỳnh quang 40 2.2.4 Phân tích nhiệt 41 2.2.5 Hiển vi Ďiện tử 42 2.2.6 Các phƣơng pháp Ďo từ 42 2.2.7 Phổ tán xạ ánh sáng Ďộng 42 2.2.8 Đốt nóng cảm ứng từ .43 2.2.9 Xác Ďịnh hiệu suất dung lƣợng mang thuốc .43 2.2.10 Q trình giải phóng thuốc in vitro .43 2.2.11 Giải phóng thuốc Ďốt nóng cảm ứng 44 2.3 Thử nghiệm sinh học 44 2.3.1 Thử nghiệm khả nhập bào Ďộc tính tế bào FOC FOCF 44 2.3.2 Xác Ďịnh phân bố hệ nano mang curcumin quan chuột 45 2.3.3 Xác Ďịnh Ďộc tính tế bào FAD, FADF, FAQ FADQ 46 2.3.4 Thí nghiệm xác Ďịnh khả Ďiều trị in vivo hệ Ďa chức mang Dox kết hợp với Ďốt nóng cảm ứng từ 46 2.4 Phƣơng pháp xử lí số liệu 48 CHƢƠNG 3: HẠT NANO Fe3O4 BỌC BẰNG OCMCS MANG CURCUMIN 49 3.1 Tổng hợp hạt nano Fe3O4 49 3.1.1 Hạt nano Fe3O4 tổng hợp theo phƣơng pháp Ďồng kết tủa thông thƣờng 49 3.1.2 Hạt nano Fe3O4 tổng hợp theo phƣơng pháp Ďồng kết tủa có hỗ trợ vi sóng 50 iii 3.2 Ảnh hƣởng hàm lƣợng curcumin lên tính chất hệ mang Curcumin (FOC1-FOC5) 55 3.3 Hệ nano mang curcumin (FOC) mang Curcumin gắn folate (FOCF) 57 3.3.1 Phổ hồng ngoại 57 3.3.2 Phổ huỳnh quang .58 3.3.3 Ảnh hiển vi Ďiện tử quét (FeSEM) 59 3.3.4 Phân tích nhiệt 60 3.3.5 Giản Ďồ nhiễu xạ tia X Ďƣờng cong từ trễ 61 3.3.6 Kết Ďốt nóng cảm ứng từ 62 3.3.7 Độ bền FOC FOCF môi trƣờng sinh lí .64 3.3.8 Q trình giải phóng thuốc in vitro 64 3.3.9 Độc tính tế bào 66 3.3.10 Phân bố sinh học 68 Kết luận chƣơng 3: 71 CHƢƠNG 4: HẠT NANO Fe3O4 BỌC BẰNG ALGINATE MANG DOXORUBICIN .72 4.1 Ảnh hƣởng nồng Ďộ alginate Ďến khả mang Dox tính chất hệ nano 72 4.1.1 Phổ hồng ngoại phổ huỳnh quang 72 4.1.2 Dung lƣợng thuốc hiệu suất mang thuốc 73 4.1.3 Phân bố kích thƣớc ảnh TEM .74 4.1.4 Giản Ďồ nhiễu xạ tia X Ďƣờng cong từ trễ 76 4.1.5 Kết Ďốt nóng cảm ứng từ 77 4.1.6 Phân tích nhiệt 80 4.1.7 Q trình giải phóng thuốc in vitro 81 4.1.8 Độc tính tế bào 83 4.2 Ảnh hƣởng lõi Fe3O4 tổng hợp vi sóng tới tính chất hệ nano .86 4.2.1 Một số Ďặc trƣng vật liệu kết Ďốt nóng cảm ứng từ .86 4.2.2 Độc tính tế bào 88 4.3 Hệ nano mang Dox gắn folate (FADF) CdTe (FADQ) 88 4.3.1 Phổ hồng ngoại 89 iv 4.3.2 Phổ huỳnh quang .89 4.3.3 Kích thƣớc hạt Zeta 91 4.3.4 Giản Ďồ XRD 91 4.3.5 Tính chất từ khả Ďốt nóng cảm ứng 92 4.3.6 Quá trình giải phóng Dox thụ Ďộng chủ Ďộng nhờ hiệu ứng Ďốt nóng cảm ứng .93 4.3.7 Độc tính tế bào 96 4.3.8 Độ bền FAD, FADF FADQ mơi trƣờng sinh lí 99 4.3.9 Kết thử nghiệm in vivo 100 Kết luận chƣơng 105 KẾT LUẬN .107 NHỮNG ĐÓNG GÓP MỚI CỦA LUẬN ÁN .109 DANH MỤC CÁC CƠNG TRÌNH SỬ DỤNG TRONG LUẬN ÁN 110 Tài liệu tham khảo 112 v DANH MỤC CÁC KÍ HIỆU, CÁC CHỮ VIẾT TẮT Alg: alginate CS% (% cell survival): số tế bào sống sót (%) Cur: Curcumin DLS (dynamic light scattering): tán xạ ánh sáng Ďộng Dox: Doxorubicin Drug: thuốc DrTGA: tốc Ďộ khối lƣợng DTA (differential thermal gravity analysis): phân tích nhiệt vi sai EE (encapsulating efficiency): hiệu suất mang thuốc EPR (enhanced permeability and retention effect): Hiệu ứng tăng tính thấm thời gian lƣu FA: mẫu Fe3O4 tổng hợp vi sóng bọc alginate nồng Ďộ mg/ml FA2D-FA10D: mẫu Fe3O4 bọc alginate nồng Ďộ khác mang doxorubicin FA2-FA10: mẫu Fe3O4 bọc alginate nồng Ďộ khác FAD: mẫu Fe3O4 tổng hợp vi sóng bọc alginate nồng Ďộ mg/ml mang doxorubicin FADF: mẫu Fe3O4 tổng hợp vi sóng bọc alginate nồng Ďộ mg/ml mang doxorubicin gắn folate FADQ: mẫu Fe3O4 tổng hợp vi sóng bọc alginate nồng Ďộ mg/ml mang doxorubicin gắn CdTe FeSEM (field emission Scanning electron mỉctoscopy): hiển vi Ďiện tử quét phát xạ trƣờng FL: tế bào ung thƣ vân tim FOC (hoặc FOC3): Fe3O4 bọc OCMCS mang curcumin với lƣợng curcumin tham gia phản ứng 60 mg FOC1-FOC5: Fe3O4 bọc OCMCS mang curcumin với lƣợng curcumin tham gia phản ứng khác (từ 20-100 mg) vi FOCF: Fe3O4 bọc OCMCS mang curcumin gắn folate (với lƣợng curcumin tham gia phản ứng 60 mg) Fol: folate FR (folate receptor): thụ thể folate H: cƣờng Ďộ từ trƣờng Hc: lực kháng từ Hela: tế bào ung thƣ cổ tử cung Hep-G2: tế bào ung thƣ gan HT-29: tế bào ung thƣ ruột kết IC50 (inhibition concentration): nồng Ďộ ức chế 50% số tế bào ILP (intrinsic loss power): công suất tổn hao nội LC (loading content): dung lƣợng thuốc LU-1: tế bào ung thƣ phổi tế bào nhỏ M (magnetization): từ Ďộ Ms (satutation magnetization): từ Ďộ bão hòa 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Ďƣợc nghiên cứu hạn chế Trên sở phân tích kể trên, chúng tơi thực luận án Nghiên cứu chế tạo đánh giá hiệu tác động hệ nano đa chức (polymer- drugFe3O4 -folate) lên tế bào ung thƣ” Mục tiêu nghiên. ..BỘ GIÁO DỤC VÀ ĐÀO TẠO VIỆN HÀN LÂM KHOA HỌC VÀ CÔNG NGHỆ VIỆT NA HỌC VIỆN KHOA HỌC VÀ CÔNG NGHỆ - LÊ THỊ THU HƢƠNG NGHIÊN CỨU CHẾ TẠO VÀ ĐÁNH GIÁ HIỆU QUẢ TÁC ĐỘNG CỦA HỆ NANO ĐA. .. quang, dung lƣợng mang thuốc, Ďộ bền, khả phân tán hệ nano Ďa chức Ďã chế tạo 3 - Xác Ďịnh khả ức chế dòng tế bào ung thƣ in vitro - Xác Ďịnh hiệu Ďiều trị ung thƣ chuột hệ nano Ďa chức Ý nghĩa

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