Department of Biotechnology, Faculty of Biological Science and Technology, University of Isfahan, Isfahan 81746-73441, Iran.
Department of Nanotechnology, Faculty of Chemistry, University of Isfahan, Isfahan 81746-73441, Iran.
Int J Pharm. 2023 Dec 15;648:123606. doi: 10.1016/j.ijpharm.2023.123606. Epub 2023 Nov 14.
In the current study, a new monoclonal antibody conjugated dual stimuli lipid-coated mesoporous silica nanoparticles (L-MSNs) platform was developed and investigated for specific co-delivery of the paclitaxel (PTX) and gemcitabine (Gem) to cancer cells and preventing their side effects during the treatment process. First, MSNs were synthesized and then coated with as-prepared pH-, and thermo-sensitive niosomes to produce L-MSNs. For this aim, Dipalmitoylphosphatidylcholine (DPPC) was used to create thermo-sensitivity, and 1, 2-Distearoyl-sn-glycerol-3-phosphoethanolamine -Citraconic Anhydride-Polyethylene Glycol (DSPE-CA-PEG) polymers were prepared and incorporated to the lipid layer for creation of pH-sensitivity. In the next step, trastuzumab as a monoclonal antibody (mAb) was conjugated to the maleimide groups of the 1, 2-Distearoyl-sn-glycerol-3-phosphoethanolamine DSPE-polyethylene glycol (PEG)-maleimide agents in the lipid bilayer via a disulfide bond. Dynamic light scattering (DLS) and zeta potential measurements, Fourier-transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), Brunauer-Emmett-Teller (BET), and scanning electron microscopy (SEM) analyses were utilized to characterize the synthesized particles before and after surface modification. The encapsulation efficiency (EE%) and loading efficiency (LE%) of the particles were also evaluated. Additionally, the drug release study and MTT assay were done to evaluate the bioactivity potential of the fabricated platforms. The results of DLS and zeta potential measurements revealed an average size of 200 nm and a neutral zeta potential of about -1 mV for mAb-L-MSNs. Also, the FTIR spectra confirmed the formation of mAb-L-MSNs. Moreover, SEM analysis showed spherical-shaped MSNs with amorphous structure confirmed by XRD analysis, and BET test revealed ∼ 820 m/g specific surface area and pore about 5 nm in size. The values of EE% and LE% of PTX were 90.3 % and 26.7 %, while these values for GEM were 89.5 % and 38.8 % in the co-loaded form, respectively. The thermo-pH-sensitivity examination showed approximately 500 nm of size increase after the change of pH and temperature from 7.4 and 37˚C to 5 and 42˚C. The release profile showed a pH-, and thermo-dependence manner, which led to about 89 % and 95 % of PTX and GEM released from the co-loaded platform at a pH of 5 and 42 °C while these values were 31.1 % and 32.2 % at pH of 7.4 and 37˚C, respectively. MTT assay data presented that when the mAb-L-co-loaded-MSNs platform containing 250 µg/mL drug was used, about 92 % of cells died in human epidermal receptors (HER2)-positive breast cancer cells (SKBR3), while just about 4 % of HER2-negative normal cells were killed. However, the growth inhibition rate of SKBR3 cells was caused by empty-mAb-L-MSNs, pure PTX and GEM combination were 9 % and 87 %, respectively. Moreover, the half inhibitory concentration (IC of the pure PTX, pure GEM, and mAb-coloaded-L-MSNs were 33, 17.6, and 6.5 µg/mL. The synergic effect of co-encapsulation of PTX and GEM in addition to trastuzumab conjugated L-MSNs was confirmed by a combinational index (CI) of 0.34. Therefore, this strategy leads to specific targeted drug delivery to cancer cells using a key-lock interaction between the trastuzumab and HER-2 receptors on the cancer cell membrane which stimuli the endocytosis of the particles to the cells followed by the destruction of the lipid layer in the acidic pH and the temperature of the lysosome, leading to enhanced release of PTX and GEM (pH of 5 and 42˚C). So, this platform can be considered a suitable carrier for cancer treatment.
在目前的研究中，开发了一种新的单克隆抗体偶联双刺激脂质包覆介孔硅纳米粒子（L-MSNs）平台，并研究了其将紫杉醇（PTX）和吉西他滨（Gem）特异性共递送至癌细胞的能力，同时防止治疗过程中的副作用。首先，合成了 MSNs，然后用预先制备的 pH 和热敏感的非离子型脂质体包覆，制得 L-MSNs。为此，使用二棕榈酰磷脂酰胆碱（DPPC）来产生热敏感性，并且制备了 1,2-二硬脂酰-sn-甘油-3-磷酸乙醇胺-Citraconic Anhydride-Polyethylene Glycol（DSPE-CA-PEG）聚合物并将其掺入脂质层中以产生 pH 敏感性。在下一步中，通过二硫键将曲妥珠单抗作为单克隆抗体（mAb）偶联到脂质双层中的 1,2-二硬脂酰-sn-甘油-3-磷酸乙醇胺二硬脂酰基聚乙二醇（PEG）-马来酰亚胺试剂的马来酰亚胺基团上。动态光散射（DLS）和zeta 电位测量、傅里叶变换红外光谱（FTIR）、X 射线衍射（XRD）、BET 和扫描电子显微镜（SEM）分析用于在表面修饰前后对合成的颗粒进行表征。还评估了颗粒的包封效率（EE％）和载药效率（LE％）。此外，进行了药物释放研究和 MTT 测定，以评估所制备平台的生物活性潜力。DLS 和 zeta 电位测量的结果表明，mAb-L-MSNs 的平均粒径为 200nm，中和 zeta 电位约为-1mV。此外，FTIR 光谱证实了 mAb-L-MSNs 的形成。此外，SEM 分析显示了具有无定形结构的球形 MSNs，XRD 分析证实了这一点，BET 测试显示了约 820m/g 的比表面积和约 5nm 的孔径。PTX 的 EE％和 LE％分别为 90.3％和 26.7％，而 GEM 的 EE％和 LE％分别为 89.5％和 38.8％。热-pH 敏感性检查表明，在 pH 值从 7.4 和 37°C 变为 5 和 42°C 以及温度从 7.4 和 37°C 变为 5 和 42°C 后，粒径增加了约 500nm。释放曲线显示出 pH 和温度依赖性，在 pH 为 5 和 42°C 时，共载药物的平台可释放约 89％和 95％的 PTX 和 GEM，而在 pH 为 7.4 和 37°C 时，这些值分别为 31.1％和 32.2％。MTT 测定数据表明，当使用含有 250μg/mL 药物的 mAb-L 共载 MSNs 平台时，HER2 阳性乳腺癌细胞（SKBR3）中的约 92％的细胞死亡，而 HER2 阴性正常细胞仅死亡约 4％。然而，SKBR3 细胞的生长抑制率是由空 mAb-L-MSNs 引起的，纯 PTX 和 GEM 的组合分别为 9％和 87％。此外，纯 PTX、纯 GEM 和 mAb 共载 L-MSNs 的半数抑制浓度（IC）分别为 33、17.6 和 6.5μg/mL。通过组合指数（CI）为 0.34 证实了 PTX 和 GEM 共包封的协同作用。因此，这种策略通过曲妥珠单抗与癌细胞膜上的 HER-2 受体之间的关键锁相互作用，实现了对癌细胞的特异性靶向药物递送，刺激了颗粒内吞到细胞中，随后在酸性 pH 和溶酶体的温度下破坏脂质层，从而增强了 PTX 和 GEM 的释放（pH 为 5 和 42°C）。因此，该平台可以被认为是一种用于癌症治疗的合适载体。
