인테그린 αvβ3을 표적으로 하는 이중 모드 프로브의 합성 및 체외 연구

나노 입자의 특성

나노입자의 입자크기는 TEM(GEOL Tokyo, Japan)으로 측정하였다. 소량의 리포솜-cRGD-Gd-Cy5.5 복합체를 초순수(pH =6.0)에 첨가하여 1mg/mL 용액으로 희석했습니다. 시료 한 방울을 코팅된 구리 그리드에 멸균 이송 피펫을 사용하여 놓고 몇 분 후에 2% 소듐 인텅스텐산을 한 방울씩 떨어뜨려 유지했습니다. 1~2분 후에 과도한 음성 염색 용액을 흡인했습니다. 그런 다음 구리 그리드를 건조시키고 관찰을 위해 80kV 투과 전자 현미경에 두었다. 약 40~50개의 나노입자를 무작위로 선택하여 형태 및 입자 크기를 관찰하고 50nm 및 100nm 크기의 이미지를 저장했습니다. 입자 크기는 NanoMeasure 소프트웨어를 사용하여 측정했으며 평균은 3회 반복 측정하여 계산했습니다.

입자 크기 분석기(Malvern, UK)를 사용하여 나노입자의 유체역학적 크기와 제타 전위를 측정했습니다. 리포솜-cRGD-Gd-Cy5.5 복합 용액 한 방울을 초순수로 희석한 다음 Eppendorf 튜브로 옮기고 초음파 세척기에 5분간 두어 입자를 고르게 분산시킵니다. 분산된 입자는 입자 크기와 제타 전위를 결정하기 위해 나노입자 크기 분석기에서 DLS로 평가되었습니다.

리포솜 cRGD-Gd-Cy5.5 복합체의 광학적 특성은 다기능 마이크로플레이트 리더(BioTek, USA)를 사용하여 특성화되었습니다. 리포솜 용액 및 리포솜-cRGD-Gd-Cy5.5 각각 2 마이크로리터를 998 μL의 인산완충식염수(PBS) 완충액에 첨가하고 석영 큐벳에 첨가하기 전에 잘 혼합했습니다. 그런 다음 다기능 마이크로플레이트 판독기를 통해 400–800nm에서 자외선 흡수를 얻었습니다. 리포솜-cRGD-Gd-Cy5.5 복합체(1.5mL) 용액을 Eppendorf 튜브에 옮기고 빈 리포솜 용액의 동일한 양을 바탕 대조군으로 사용했습니다. 형광 이미징은 작은 동물 형광 이미징 시스템을 사용하여 수행되었습니다.

리포솜 표면 아미노기의 변형은 FT-IR 분광기(Nicolet-5700, USA)를 이용하여 확인하였다. UV-vis 흡수 스펙트럼을 얻었다(UV 2550, Shimadzu, Japan). 0.1 mg/mL 농도의 리포솜 cRGD-Gd-Cy5.5가 측정에 사용되었습니다.

이완율 측정

cRGD-변형된 Gd-로딩된 리포솜 형광 프로브 샘플을 탈이온수로 희석하여 다음 Gd 농도를 얻었다:0.18, 0.1275, 0.085, 0.0425, 0.017mM. 희석된 각 시료 5ml를 스크류 캡이 있는 바이알에 넣은 후, 바이알을 농도 순서에 따라 다기능 튜브 랙에 고정하였다. cRGD-modified Gd-loaded liposomal 형광 프로브 샘플은 다양한 농도의 프로브에 대한 T1 강조 이미지를 얻기 위해 머리와 목 코일을 통한 MR 스캐닝을 거쳤습니다. T1 강조 이미지에 사용된 스캔 시퀀스는 MOLLI(Modified Look-Locker Inversion Recovery) 시퀀스로 매개변수가 다음과 같이 설정되었습니다. 반복 시간(TR) =5.8ms, 에코 시간(TE) =3.66ms, 반전 복구 시간(TI) =16–3200ms, 스캐닝 두께 =5 mm. 스캔이 완료된 후 유사 색상 맵을 얻었습니다. 0.3cm ² 지도의 각 샘플 이미지에 관심 영역을 표시하고 해당 T1 값을 읽습니다.

세포 배양 및 세포독성 분석

A549 인간 폐 선암종 세포, SUNE-1-5-8F 인간 비인두 암종 세포, 인간 제대 정맥 내피 세포(HUVEC) 및 MCF-10A 인간 유방 상피 세포는 American Type Culture Collection(ATCC, Manassas, VA)에서 입수했습니다. . 세포는 10% 소태아 혈청(Euroclone-Lonza), 100units/mL 페니실린, 100μg/mL 스트렙토마이신 및 2mM 글루타민을 5% CO2에서 포함하는 완전한 1640 배지(Euroclone-Lonza)에서 배양되었습니다. /서브> 37°C에서.

CCK-8 방법을 사용하여 정상 상피 세포 및 종양 세포를 포함한 다른 세포에 대한 분자 프로브의 세포 독성을 조사했습니다. A549 세포, 5-8F 세포, HUVEC 및 MCF-10A 세포를 96웰 플레이트에 5 × 10 ³ 의 밀도로 시딩했습니다. 세포/웰 및 밤새 배양하였다. The adherent cells were then incubated with 100 μL of fresh 1640 medium containing cRGD-Gd-Cy5.5 of various Gd concentrations (0, 50, 100, 200, and 400 μM) for 24 h. Subsequently, the cells were washed three times with PBS and then incubated with 100 μL of Dulbecco’s modified Eagle medium (DMEM) containing no fetal bovine serum (FBS). After 10% Cell Counting Kit-8 reagent (Dojindo, Japan) was added to each well, the sample was incubated for 10 h. The absorbance at 450 nm of each well was then measured using an ELISA microplate reader (Multiskan MK3, Thermo Scientific, Logan, UT). Cells treated with PBS only were employed as the control group. For each sample, five parallel wells were analyzed to obtain the mean and standard deviation.

Immunofluorescence Staining and Flow Cytometry Assay of Integrin αvβ3

A total of 20,000 A549, 5-8F, and MCF-10A cells each were seeded in confocal plates (Thermo Scientific) and cultured for approximately 24 h when the cells successfully grew on the slides. The cells were washed three times with PBS, fixed with 4% paraformaldehyde (Boster) at room temperature for 30 min, and then washed three times (10 min each) in PBS. The cells were then incubated in 3% bovine serum albumin (PBST) for 30 min to block non-specific antibody binding and washed three times with PBS. The fixed cells were incubated with anti-integrin αvβ3 monoclonal antibody (1:500; Abcam, Cambridge, UK) at 4 °C overnight and then incubated with anti-mouse fluorescent secondary antibody (1:500) (Abcam) for 1 h. 4′,6-Diamidino-2-phenylindole (DAPI; 1:1000; Sigma) was used to stain the nucleus. A laser confocal scanning microscope (Nikon A1, Tokyo, Japan) was used to detect the green fluorescence signal from integrin αvβ3 and the DAPI signal from nuclei.

The integrin αvβ3 expression levels in different cell lines were quantified using flow cytometry. In brief, three types of cells were collected:A549, SUNE-1-5-8F, and MCF-10A, which were washed in PBS containing 1% bovine serum albumin (BSA). After being sealed with the PBS containing 5% BSA, the cells were cultivated in VNR-1 antibody (Abcam, Cambridge, MA, USA) at density 2 μg/1 × 10 ⁶ and 4 °C for 30 min. The secondary antibody was DyLight 488 goat anti-mouse IgG (H+L) (ab96879), diluted 1/500, and at 22 °C for 30 min. Quantitative assays were conducted on a flow cytometer (Gallios, Beckman Coulter, USA) at excitation wavelength 488 nm and emission wavelength 530 nm. Each time, at least 1 × 10 ⁵ cells (n  = 3) were collected.

Binding Assay of the Molecular Probe to Integrin αvβ3

A549, SUNE-1-5-8F, and MCF-10A cells were inoculated into confocal plates. The cells were incubated at 37 °C for 1 h with the molecular probe of the same Gd concentration after the cells reached the logarithmic growth phase. The cells were then washed three times in PBS and placed under a laser confocal scanning microscope for observation.

In Vitro MR Imaging and Fluorescence Imaging of Tumor Cells

To verify the tumor cell-targeting ability of the cRGD-modified Gd-loaded liposomal fluorescent probe, the probe was incubated with A549 cells and then examined using MR imaging and a small animal fluorescence imaging system. A549 cells were seeded in six-well plates with 2 mL of 1640 medium containing 10% FBS and 1% penicillin–streptomycin in each well and then incubated at 37 °C and 5% CO₂ . In the experimental groups, cRGD-Gd-Cy5.5 was incubated with A549 cells for 2 h with Gd concentrations of 0, 0.005, 0.01, 0.02, 0.04, and 0.08 mM. The cells of the control groups were incubated with the non-targeted gadolinium contrast agent (Gd-Cy5.5) for 2 h at the same Gd concentrations as the experimental groups. The cells were then washed with PBS, trypsinized, centrifuged, and finally placed in glass tubes containing 1 mL of PBS (with 0.5% agarose) for MR imaging. Specific imaging parameters were described above. A small animal live imaging system (IVIS Lumina XRMS Series III, MA, USA) was used for fluorescence imaging of the A549 cells.

In Vivo MR and Fluorescence Imaging

To validate whether CRGD-Gd-Cy5.5 had tumor targetability in vivo in animals, we conducted animal experiments following the Guide for Care and Use of Laboratory Animals, released by the Animals Ethics Committee of Laboratory Animal Center, Guangxi Medical University. We successfully established a Balb/c nude mice nasopharyngeal carcinoma subcutaneous xenografting tumor model for magnetic resonance/fluorescent scanning. A 3.0-T MRI scanner containing 40-mm-diameter mouse volume coils (Discovery 750, GE, Germany) was used for T1-weighted imaging, operated at field of view = 80 × 80 mm, repetition time = 742 ms, echo time = 69 ms, and layer thickness = 2.0 mm. The nude mice were divided into two groups (n  = 3), including a test group and a receptor-blocked group. All mice were injected via tail vein with CRGD-Gd-Cy5.5 (0.05 mmol Gd/kg). Each mouse was scanned at four time points:before injection, 30 min, 2 h, and 6 h after injection. In the receptor-blocked group, 1 h before injection with CRGD-Gd-Cy5.5, each mouse was injected via the tail vein with free CRGD polypeptide (10 mg) and was MRI-scanned at the same four time points. The fluorescent images were photographed by a fluorescence imaging system (Bruker, USA). The grouping, drug injection, and scanning time points were all the same as described above. During the imaging, the mice (n  = 3) were anesthetized using a gas mixture of oxygen and isoflurane. The nude mice were kept at heart rate 60–120/min and respiratory rate at 20–40/min. Finally, direct visual comparison of tumor images was conducted by two experienced radiologists.