雙胜肽接枝奈米劑型應用於抗癌藥物遞送

Abstract

標靶遞送近年來在醫療領域被廣泛的研究，尤其以奈米顆粒遞送系統為一大發展方向。在形成奈米顆粒的材料中，聚乳酸-甘醇酸 (Poly (lactide-co-glycolide), PLGA)所形成之奈米顆粒具有高穩定性與低毒性的優勢，在標靶遞送的研究領域備受矚目。 本實驗以聚乳酸-甘醇酸 (Poly(lactide-co-glycolide), PLGA)與聚乙二醇二胺 (Poly (ethylene glycol) bis (amine), PEG diamine)合成PLGA-PEG共聚物，該共聚物經過核磁共振 (H1-NMR)、傅立葉轉換紅外線光譜儀確認化學結構與接枝率。接著，將兩種不同的胜肽－具有表皮生長因子受體標靶能力的NR7與細胞穿透肽TAT分別接枝在PLGA-PEG共聚物上。在細胞吞噬試驗中，將單一胜肽接枝或雙胜肽混合的PLGA-PEG以溶媒揮發法製備奈米顆粒，分別對於表皮生長因子受體過度表現的細胞株(SKOV3)與無過度表現的細胞株(MCF-7)進行試驗並比較。藥物包覆方面，以無胜肽、單一胜肽接枝或雙胜肽混合的PLGA-PEG以溶媒揮發法分別製備包覆歐洲紫杉醇或薑黃素的奈米顆粒，形成包覆藥物之無胜肽接枝奈米顆粒(NPs)、單胜肽接枝奈米顆粒(NR7-NPs、TAT-NPs)與雙胜肽接枝奈米顆粒(NR7TAT-NPs)，並監測其粒徑、表面電位、包覆率與載藥率，並進一步進行含藥奈米顆粒的體外釋放試驗，確認藥物於pH 7.4及pH 4.0溶媒中的控釋功能。此外，本實驗以MTT試驗驗證胜肽接枝奈米顆粒的細胞吞噬效果，針對EGFR過度表現或無過度表現的細胞株進行歐洲紫杉醇與薑黃素單一藥物遞送試驗或共同藥物遞送試驗。另一方面，為了驗證奈米顆粒能提高生體可用率的效果，本實驗進行各劑型的藥物動力學試驗，監測大鼠的血中薑黃素與歐洲紫杉醇濃度，並與純藥注射的組別比較。 實驗結果顯示，PLGA與PEG diamine接枝率可達292.5±4.2%，而在NR7-FITC與TAT-TAMRA的接枝反應中，在pH8.5環境中接枝率可分別達48.9±5.9%與87.5±8.5%。在EGFR過度表現細胞株的吞噬試驗中，接枝NR7的奈米顆粒吞噬程度為33.47±3.95%，接枝TAT的奈米顆粒則為57.04±3.80%，雙胜肽混和的奈米顆粒吞噬程度為78.51±4.48%；在EGFR無過度表現的細胞株方面，接枝NR7的奈米顆粒吞噬程度為11.04±3.57%，與對照組無差異，接枝TAT的奈米顆粒則為90.36±4.28%，雙胜肽混和的奈米顆粒吞噬程度為84.93±9.77%，與接枝TAT的奈米顆粒無顯著差異。 奈米顆粒製備的部分，NPs-CUR粒徑為154.6±17.1 nm、PDI為0.11±0.05、表面電位為18.0±13.8 mV、包覆率為50.7±6.3%、載藥率為6.9±0.3%；NR7-NPs-CUR粒徑為137.0±4.4 nm、PDI為0.09±0.01、表面電位為-12.7±2.1、包覆率為56.0±4.8%、載藥率為7.4±0.3%；TAT-NPs-CUR粒徑為131.0±6.0 nm、PDI為0.05±0.02、表面電位為-12.8±2.10 mV、包覆率為54.8±4.3%、載藥率為7.2±0.2%；NR7TAT-NPs-CUR粒徑為129.6±12.1 nm、PDI為0.14±0.07、表面電位為-7.9±1.3 mV、包覆率為58.6±2.7%、載藥率為7.5±0.2%；NPs-DTX粒徑為196.5±7.1 nm、PDI為0.25±0.5、表面電位為15.8±4.0 mV、包覆率為79.6±5.2%、載藥率為13.0±0.3%；NR7-NPs-DTX粒徑為199.1±3.4 nm、PDI為0.25±0.3、表面電位為-15.1±2.0 mV、包覆率為80.6±8.7%、載藥率為12.6±0.9%；TAT-NPs-DTX粒徑為163.0±16.4 nm、PDI為0.16±0.5、表面電位為-13.1±0.9 mV、包覆率為69.7±6.0%、載藥率為11.9±0.6%；NR7TAT-NPs-DTX粒徑為174.7±1.2 nm、PDI為0.18±0.3、表面電位為-14.6±1.2 mV、包覆率為83.9±7.1%、載藥率為13.1±0.5%。在細胞毒性試驗中，Free CUR對SKOV3的IC50為3.82±0.34 µg/mL、NPs-CUR為3.56±0.18 µg/mL、NR7-NPs-CUR為2.96±0.11 µg/mL、TAT-NPs-CUR為1.87±0.63 µg/mL，NR7TAT-NPs-CUR則為2.37±0.32 µg/mL，NR7-NPs-CUR的IC50與NPs-CUR及Free CUR有顯著差異，NR7TAT-NPs-CUR及TAT-NPs-CUR與其他三組劑型皆有顯著差異；Free DTX對SKOV3的IC50為14.85±3.91 ng/mL、NPs-DTX為5.46±2.16 ng/mL、NR7-NPs-DTX為1.44±0.57 ng/mL、TAT-NPs-DTX為3.44±1.02 ng/mL，NR7TAT-NPs-DTX則為0.68±0.04 ng/mL，TAT-NPs-CUR對NPs-DTX無顯著差異，NR7-NPs-DTX與NR7TAT-NPs-DTX對NPs-DTX則有顯著差異。在Co-delivery部分，NR7TAT-NPs-DTX配合NR7TAT-NPs-CUR亦表現最佳的毒殺效果。 在藥物動力學試驗方面，NPs-CUR與NR7-NPs-CUR及NR7TAT-NPs-CUR的藥物濃度曲線下面積無顯著差異 (p>0.05)，且皆大於注射薑黃素純藥組別的十五倍至三十倍；NPs-DTX與NR7-NPs-DTX及NR7TAT-NPs-DTX之藥物濃度曲線下面積也無顯著差異 (p>0.05)，並大於注射歐洲紫杉醇純藥組別五倍至七倍。

In recent years, target delivery are being extensively researched. Specially, nanoparticles delivery system is a major development field. Among the material formed nsnoparticles, Poly (lactide-co-glycolide) (PLGA) has the advantages of high stability and low toxicity,getting much attention in target delivery research. In this study, PLGA-PEG copolymer was synthesized with PLGA and PEG diamine. The copolymer was checked by nuclear magnetic resonance (H1-NMR) and fourier transform infrared spectrometer (FTIR) to confirm the chemical structure and conjugation ratio. Next, two different peptides, NR7, with epidermal growth factor receptor targeting ability, and TAT, one fo cell penetrating peptide, were conjugated to the PLGA-PEG copolymer. In cellular uptake assay, nanoparticles were prepared by solvent evaporation method with single peptide or double peptide conjugation. The SKOV3 cell lines, which were overexpressed in epidermal growth factor receptor and the non-overexpressed MCF-7 cell lines were tested and compared. Then, docetaxel and curcumin loaded nanoparticles were prepared by solvent evaporation methods and form the peptides-free (NPs), single peptide (NR7-NPs、TAT-NPs) and double peptides (NR7TAT-NPs) conjugated drug loaded nanoparticles, and the particle size, surface potential, entrapment efficiency and drug loading were monitored. Nanoparticles also were confirm the controlled release function in pH7.4 and pH4.0 by in vitro release assay. In addition, the cellular uptake of peptide-conjugated nanoparticles was verified by MTT assay. The docetaxel and curcumin single and co-drug delivery were test. Final, pharmacokinetic test were carried out to comfirm that nanoparticles can improve the bioavailability of the drug. The results showed that the conjugated ratio of PLGA and PEG diamine was 292.5±4.2%. The conjugated ratio at pH 8.5 of NR7 and TAT were 48.9±5.9% and 87.5±8.5%, respectively. In the cellular uptake of EGFR overexpressing cell lines, the uptake ratio of NR7-NPs was 33.47±3.95%, TAT-NPs was 57.04±3.80% and NR7TAT-NPs was 78.51±4.48%. In EGFR nonoverexpressing cell lines, the uptake ratio of NR7-NPs was 11.04±3.57%, TAT-NPs was 90.36±4.28% and NR7TAT-NPs was 84.93±9.77%. The particle size、PDI and zeta potential of NPs-CUR were 154.6±17.1 nm、0.11±0.05 and 18.0±13.8 mV, the entrapment efficiency(EE) and drug loading(DL) of NPs-CUR were 50.7±6.3% and 6.9±0.3%；the particle size、PDI and zeta potential of NR7-NPs-CUR were 137.0±4.4 nm、0.09±0.01 and -12.7±2.1 mV, the EE and DL of NR7-NPs-CUR were 56.0±4.8% and 7.4±0.3%；the particle size、PDI and zeta potential of TAT-NPs-CUR were 131.0±6.0 nm、0.05±0.02 and -12.8±2.10 mV, the EE and DL of TAT-NPs-CUR were 54.8±4.3% and 7.2±0.2%；the particle size、PDI and zeta potential of NR7TAT-NPs-CUR were 129.6±12.1 nm、0.14±0.07 and -7.9±1.3 mV, the EE and DL of NR7TAT-NPs-CUR were 58.6±2.7% and 7.5±0.2%；the particle size、PDI and zeta potential of NPs-DTX were 196.5±7.1 nm、0.25±0.5 and 15.8±4.0 mV, the EE and DL of NPs-DTX were 79.6±5.2% and 13.0±0.3%；the particle size、PDI and zeta potential of NR7-NPs-DTX were 199.1±3.4 nm、0.25±0.3 and -15.1±2.0 mV, the EE and DL of NR7-NPs-DTX were 80.6±8.7% and 12.6±0.9%；the particle size、PDI and zeta potential of TAT-NPs-DTX were 163.0±16.4 nm、0.16±0.5 and -13.1±0.9 mV, the EE and DL of TAT-NPs-DTX were 69.7±6.0% and 11.9±0.6%；the particle size、PDI and zeta potential of NR7TAT-NPs-DTX were 174.7±1.2 nm、0.18±0.3 and -14.6±1.2 mV, the EE and DL of NR7TAT-NPs-DTX were 83.9±7.1% and 13.1±0.5%. In the cytotoxicity assay, the IC50 of Free CUR to SKOV3 was 3.82±0.34 µg/mL, NPs-CUR was 3.56±0.18 µg/mL, NR7-NPs-CUR was 2.96±0.11 µg/mL, TAT-NPs-CUR was 1.87±0.63 µg/mL, and NR7TAT-NPs-CUR was 2.37±0.32 µg/mL, showing better delivery effect than the other groups (p<0.05). The IC50 of Free DTX to SKOV3 was 14.85±3.91 ng/mL, NPs-DTX was 5.46±2.16 ng/mL, NR7-NPs-DTX was 1.44±0.57 ng/mL, TAT-NPs-DTX was 3.44±1.02 ng/mL, and NR7TAT-NPs-DTX was 0.68±0.04 ng/mL. There was no significant different between the IC50 of TAT-NPs-DTX and NPs-DTX, however, the IC50 of NR7-NPs-DTX and NR7TAT-NPs-DTX were significantly different for NPs-DTX. In co-delivery test, NR7TAT-NPs-CUR and NR7TAT-NPs-DTX also showed better cytotoxicity than free dug and single peptide NPs in SKOV3 cell. In the pharmacokinetic test, the AUC of different group of NPs-CUR、NR7-NPs-CUR and NR7TAT-NPs-CUR were not significantly different (p > 0.05), and all were better than free curcumin by fifteen to thirty times; the AUC of NPs-DTX、NR7-NPs-DTX and NR7TAT-NPs-DTX also were not significantly different (p > 0.05) and all were better than free docetaxel by five to seven times.