항-Epcam 앱타머(Syl3c)-독소루비신의 표적 전달을 위한 기능화된 리포솜:C26 결장암을 보유한 마우스의 시험관 내 및 생체 내 항종양 연구

재료 및 방법

자료

5'-Amine-anti-EpCAM DNA 앱타머(5'-CACTACAGAGGTTGCGTCTGTCCCACGTGTCATGGGGGGTTGGCCTG-3')(SYL3C)는 BIONEER(biotechnology company, Daejeon, South Korea)에서 구입했습니다. DSPE-mPEG₂₀₀₀ -COOH는 Avanti Polar Lipids(Alabaster, AL)에서 구입했습니다. Dowex®, 1-에틸-3-(3-디메틸아미노프로필) 카르보디이미드(EDC), N-히드록시숙신이미드(NHS), 페니실린 스트렙토마이신 및 DAPI가 포함된 Fluoroshield™는 Sigma-Aldrich(St. Louis, MO)에서 구입했습니다. 상업적으로 이용 가능한 caelyx®는 Behestan Darou Company(이란 테헤란)에서 구입했습니다.

DSPE-mPEG의 활용₂₀₀₀ 압타머로

Anti-EpCAM 앱타머는 DSPE-mPEG₂₀₀₀에 연결되었습니다. DSPE-mPEG₂₀₀₀의 카르복실기(–COOH)에 항-EpCAM 앱타머의 1차 아민(-NH2)의 공유 결합을 통해 (그림 1). 접합은 EDC/NHS 커플링 화학을 통해 수행되었습니다[51]. 간단히 말해서 DSPE-mPEG₂₀₀₀ 2-(N-모르폴리노)에탄설폰산(MES) 완충액(pH 6.5)에 분산시키고 EDC/NHS 400mM EDC 및 100mM NHS를 분산액에 첨가하였다. 분산액을 15분 동안 교반하여 지질의 카르복실기를 활성화시켰다. 그런 다음, 항-EpCAM 앱타머를 분산액에 첨가하고 암실 상온에서 다음 2시간 동안 교반하였다. lipid:anti-EpCAM aptamer의 몰비는 1:1이었고 EDC/NHS의 몰비는 지질의 10배였습니다.

DSPE-mPEG-Anti-EpCAM Aptamer를 사용한 Caelyx® 수정

ED-lip은 post-insertion 방법으로 합성하였다. 삽입 후를 수행하기 위해 DSPE-mPEG-항-EpCAM 앱타머 미셀을 60°C에서 30분 동안 1ml의 caelyx®에 첨가했습니다. DSPE-mPEG-EpCAM 앱타머의 양은 Bartlett phosphate assay[52]에 따라 결정되었다. 약 10 ¹⁴ 인 caelyx® 밀리리터당 리포솜의 대략적인 수를 기준으로 합니다. , DSPE-mPEG-항-EpCAM의 부피는 각 리포솜당 10 압타머에 도달하도록 조정되었습니다[36]. 삽입 후 확인에 사용되는 아가로스 겔 전기영동 [39].

이화학적 특성화

입자 크기, 다분산 지수(PDI) 및 표면 전하는 DLS(Dynamic Light Scattering instrument)(Nano-ZS; Malvern, UK)에 의해 결정되었습니다. 유리 DOX를 제거하기 위해 리포솜을 Dowex® 수지와 혼합하고 60분 동안 회전시키고 Dowex®를 제거하기 위해 Poly-Prep 컬럼(Bio-Rad Laboratories Inc.)을 통과했습니다[53]. 리포솜 제형에서 DOX의 양은 LS-45 형광 분광광도계(Perkin-Elmer, UK)를 사용하여 결정되었습니다(여기:방출 485:590 nm).

출시 연구

DOX의 방출을 평가하기 위해 특정 시간 간격(0, 1, 2, 4, 6, 12 및 24시간), 샘플을 채취했습니다. Dowex® 수지로 유리 DOX를 제거한 후 리포솜에 남아 있는 약물의 양을 형광 분광 광도계로 측정하고 방출 비율을 계산했습니다[39].

세포 배양

C26 결장암 및 차이니즈 햄스터 난소(CHO-K1) 세포주는 이란 파스퇴르 연구소에서 구입했습니다. 세포주는 Gibco(Thermos Fisher Scientific, USA)로부터 입수한 10% FBS 및 100IU/ml 페니실린 및 100mg/ml 스트렙토마이신이 보충된 RPMI1640 배지에서 배양되었다. 세포를 37 ^° 에서 배양했습니다. C 5% CO₂ 95% 공기 가습 대기.

세포 상호작용 및 세포 흡수 분석

제형의 세포 상호작용 및 세포 흡수를 각각 4°C 및 37°C에서 평가했습니다. Two cell lines, CHO-K1 and C26, were selected in this test. The cells seeded in each well of 12-well plates (2.5 × 10 ⁵ per well). After overnight incubation in 37 °C, treatments added to the cells and plates were placed at 4 °C and 37 °C and incubate for another 3 h. Then cells washed with PBS, and trypsinized. The fluorescence intensity for DOX was determined using flow cytometry (BD FACSCalibur cytometer). The data were analyzed with FlowJo version 7.0 software.

Fluorescent Microscopy Evaluation

The number of 1 × 10 ⁶ cells per well C26 Cells were seeded into 6-well plates in which sterile microscopic cover glass were already inserted. After overnight incubation in 37 °C and 5% humidity, cells were treated with free DOX, Caelyx® and ED-lip for 24 h for complete cell uptake [54]. Then cells washed with PBS and fixed with 4% formaldehyde. Cover glasses stained with Fluoroshield™ with DAPI and were mounted on the glass slides. Treatments were performed in triplicate. From each slide, six zones were selected under × 200 magnification field. Intrinsic fluorescent of DOX was used for evaluation of drug cell uptake. Scaling was performed based on the percentages of cells which shown DOX cell uptake in each microscopic filed:

1:0–20%, 2:20–40%, 3:40–60%, 4:60–80%, and 5:80–100%

Evaluation of Cytotoxicity

The IC50 values of free DOX, caelyx®, and ED-lip were determined by MTT assay. In order to do this, CHO-K1 and C26 cells were seeded at density of 5 × 10 ³ cells per well in 96-well plates at 37 °C. After overnight incubation liposomal formulations and free DOX solution were serially diluted in FBS-free medium and added to cell cultures and incubated 1, 3, and 6 h at 37 °C. Then, cells were washed and allowed to incubate 72 h. The optical densities (ODs) were measured using a spectrometric absorbance of 570 nm against a background of 630 nm on Stat-Fax 2100 microplate reader (Awareness Technology Inc. USA). Then the IC50 values were calculated.

Animal Study

Female BALB/c mice (4–6 weeks, 18–20 g) were kept in separate cages at 22 ± 2 °C and maintained on standard pellet diet and water ad libitum. Intraperitoneal (i.p) injection of ketamine and xylazine (100 mg/kg ketamine and 10 mg/kg xylazine) used to anesthetize the animals [55]. The number of 3 × 10 ⁵ C26 cells per mouse in 60 μl PBS injected at the right flank, subcutaneously. Two weeks after inoculation when tumor sizes grew about 5 mm ³ , mice were randomly divided into 3 groups (n =3 for biodistribution and n =5 for antitumor study mice per group). All of the experimental protocols were approved by the Mashhad University of Medical Sciences committee for animal ethics and were performed according to the international rules considering the animal rights.

Biodistribution and Pharmacokinetic Studies

Fourteen days after tumor inoculation, mice were treated with dose of 10 mg/kg of caelyx® and ED-lip intravenously (i.v.) via the tail vain. Control group received 200 μl PBS solution. At certain time-intervals (3, 12, 24, 48, and 72 h) post-injection mice were euthanized and blood samples and tissue samples (liver, spleen, kidney, lung, heart, and tumor) were collected. Then, the concentration of doxorubicin in each sample measured based on fluorescent intensity of each samples using LS-45 fluorescence spectrophotometer (Perkin-Elmer, UK). Doxorubicin concentration of each sample was measured and non-compartmental analysis of the pharmacokinetic parameters were calculated from blood concentration vs. time profiles. Then the parameters including under the concentration-time curve (AUC) and area under the first moment curve (AUMC), half-life (t _1/2 ), volume of distribution (V _d ), C _최대 , T _max,, mean residence time (MRT), and clearance (Cl) were calculated.

In Vivo Antitumor Activity

In order to evaluate antitumor activity, 10 days after tumor inoculation, mice with palpable tumor size were received single i.v. dose of 10 mg/kg Caelyx® and ED-lip. PBS injected in mice which considered as negative control. The parameters including time to reach the endpoint (TTE), percentage of tumor growth delay (TGD), median survival time (MST), and survival were determined. During the study, mice were observed for health and body weight changes. The tumor volume was also measured using a digital caliper and calculated as follows:

Considering ethical aspects, mice were removed in case tumor growth was> 1000 mm ³ , or> 20% weight loss or sign of weakness was observed.

Statistical Analysis

Data were analyzed using GraphPad Prism 6.0 (GraphPad software, Inc., San Diego, CA, USA). Data were demonstrated as mean ± SEM of at least three independent experiments. The t test was used in order to evaluate the results of release study, flow cytometry, and biodistribution of the formulations. ANOVA was employed to evaluate the results of fluorescent microcopy and tumor volumes. The Kaplan–Meier method used to calculate the survival parameters include TTE, MST and TGD%. 피 <0.05 was considered statistically significant.