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  目前，通過化學(xué)療法治療癌癥患者是臨床研究熱點(diǎn)之一，但是由于抗癌藥物的副作用和多次藥物后的免疫力現(xiàn)象導(dǎo)致化學(xué)療法在臨床醫(yī)學(xué)上受到極大限制。由于腫瘤細(xì)胞擴(kuò)散較快，單純的基因治療難以達(dá)到長期抑制的作用。因此聯(lián)合基因和藥物控釋治療方法，抑制腫瘤細(xì)胞并避免多次服用藥物引起的藥物免疫性，目前已成為新的研究方向。這種方法對載體材料提出了較高要求，需要同時負(fù)載抗癌藥物和目標(biāo)基因并且互不干擾，有效地將藥物和基因輸送到靶細(xì)胞并進(jìn)行釋放和表達(dá)。

  這項(xiàng)工作通過點(diǎn)擊化學(xué)反應(yīng)和酰胺化反應(yīng)，合成以殼聚糖 (Cs) 為主鏈、聚酰胺-胺樹枝狀分子 (polyamidoamine, PAMAM) 基元和脫氧膽酸 (DCA) 為側(cè)鏈的兩親性梳形聚合物 (PAMAM-Cs-DCA) (Scheme 1)。¹H NMR和FTIR表征確定了產(chǎn)物的結(jié)構(gòu) (Figure 1)。

Scheme 1. (A) Schematic representation of co-delivering hydrophobic drug and pDNA by PAMAM-Cs-DCA. Synthesis routes of (B) 6-N₃-Cs and (C) PAMAM-Cs-DCA.

Figure 1. (a) FT-IR spectra of propargyl focal point PAMAM dendron (G=3), 6-N₃-Cs-DCA and PAMAM-Cs-DCA. (b) ¹H NMR spectra of PAMAM dendron (G=3), 6-N₃-Cs-DCA₇, and PAMAM₁₄-Cs-DCA₇ in DMSO-d₆.

  PAMAM-Cs-DCA在水中自組裝形成以PAMAM和Cs為親水外殼、DCA為疏水內(nèi)核的納米膠束，通過熒光光譜、動態(tài)光散射(DLS)和TEM測試，研究了PAMAM-Cs-DCA的膠束化行為 (Figure 2)。

Figure 2. Intensity ratios (I₁/I₃) from pyrene emission spectra in PBS buffer (pH 7.4) against the logarithm of concentration of (a) PAMAM₁₂-Cs-DCA₁₁ NPs or (b) PAMAM₁₄-Cs-DCA₇. TEM images and size distributions obtained from DLS of the NPs from (c) ) PAMAM₁₂-Cs-DCA₁₁ and (d) PAMAM₁₂-Cs-DCA₁₁-DOX.

  以阿霉素為模型藥物，考察了PAMAM-Cs-DCA納米膠束的藥物負(fù)載能力和藥物釋放行為，藥物負(fù)載率隨著DCA取代度的增加而增加，在整個藥物釋放過程中載藥膠束PAMAM-Cs-DCA-DOX的藥物釋放速率都保持緩慢而穩(wěn)定，并隨著pH值的降低而變快 (Figure 3)。

Figure 3. In vitro release profiles of entrapped DOX from PAMAM₁₂-Cs-DCA₁₁ NPs and PAMAM₁₄-Cs-DCA₇ NPs in phosphate buffer (pH 7.4 or 5.2) at 37 ℃. The inset is an enlarged image for showing the initial release. (n=3, mean ± standard deviation)

  凝膠電泳實(shí)驗(yàn)表明當(dāng)N/P值大于等于1:1時，PAMAM-Cs-DCA和負(fù)載藥物的PAMAM-Cs-DCA-DOX都能通過親水殼PAMAM基元包裹pDNA，并完全阻滯pDNA在凝膠電泳中的遷移。DLS和Zeta電位測試表明，PAMAM-Cs-DCA和PAMAM-Cs-DCA-DOX能和pDNA形成粒徑為200-300 nm的球形復(fù)合物，表面電荷為正值。MTT測試表明PAMAM-Cs-DCA和PAMAM-Cs-DCA-DOX在293T細(xì)胞中具有較低的細(xì)胞毒性，遠(yuǎn)低于PEI(25 KDa)的細(xì)胞毒性；負(fù)載藥物的PAMAM-Cs-DCA-DOX的細(xì)胞毒性稍大于PAMAM-Cs-DCA。PAMAM-Cs-DCA / pDNA復(fù)合物和PAMAM-Cs-DCA-DOX / pDNA復(fù)合物都能有效地將pDNA轉(zhuǎn)進(jìn)293T細(xì)胞中，轉(zhuǎn)染效率隨N/P值的增加而增加；并且負(fù)載阿霉素后，復(fù)合物的轉(zhuǎn)染效率稍微降低 (Figure 4和Figure 5)。

Figure 4. Transfection efficiency of PAMAM₁₂-Cs-DCA₁₁ NPs/pDNA, PAMAM₁₄-Cs-DCA₇ NPs/pDNA, PAMAM₁₂-Cs-DCA₁₁-DOX NPs/pDNA and PAMAM₁₄-Cs-DCA₇ -DOX NPs/pDNA complexes in 293T cells at various N/P ratios using PEI/DNA complexes as the control. (*p<0.05, n=3, mean ± standard deviation)

Figure 5. Representative fluorescence images of 293T cells transfected by PAMAM₁₂-Cs-DCA₁₁NPs/pDNA complex (a: N/P 5:1; b: N/P 10:1; c: N/P 20:1), PAMAM₁₂-Cs-DCA₁₁-DOX NPs/pDNA complex (d: N/P 5:1; e: N/P 10:1; f: N/P 20:1), PEI/pDNA complex (g: N/P 10) and native pDNA (h) (AI and BI: fluorescence field images; AII and BII: bright field images).

  以上相關(guān)成果以“Cationic nanoparticles self-assembled from amphiphilic chitosan derivatives containing poly(amidoamine) dendrons and deoxycholic acid as a vector for co-delivery of doxorubicin and gene”為題在 Carbohydrate Polymers (2021，Article 117706) [SCI impact factor: 7.182] 上發(fā)表。論文的共同第一作者為陳珊珊博士生和鄧俊杰博士，通訊作者為中山大學(xué)材料科學(xué)與工程學(xué)院張黎明教授。該研究得到國家自然科學(xué)基金、廣東省自然科學(xué)基金和聚合物復(fù)合材料及功能材料教育部重點(diǎn)實(shí)驗(yàn)室的資助與支持。

  論文鏈接: https://www.sciencedirect.com/science/article/abs/pii/S014486172100093X