Combined integrin αvß3 and lactoferrin receptor targeted docetaxel liposomes enhance the brain targeting effect and anti-glioma effect.

1. 논문 제목: Combined integrin α_vß₃ and lactoferrin receptor targeted docetaxel liposomes enhance the brain targeting effect and anti-glioma effect.

2. 저자명: Na Qi, Shangqian Zhang, Xiantai Zhou, Wenjuan Duan, Duan Gao, Jianfang Feng and Aimin Li

3. 출처 및 게재 (발행일): Journal of Nanobiotechnology, 23 December 2021

4. DOI: https://doi.org/10.1186/s12951-021-01180-0

5. Abstract:

The integrin αvβ3 receptor and Lactoferrin receptor (LfR) are over-expressed in both cerebral microvascular endothelial cells and glioma cells. RGD tripeptide and Lf can specifically bind with integrin α_vβ₃ receptor and LfR, respectively. In our study, RGD and Lf dual-modified liposomes loaded with docetaxel (DTX) were designed to enhance the brain targeting effect and treatment of glioma. Our in vitro studies have shown that RGD-Lf-LP can significantly enhance the cellular uptake of U87 MG cells and human cerebral microvascular endothelial cells (hCMEC/D3) when compared to RGD modified liposomes (RGD-LP) and Lf modified liposomes (Lf-LP). Free RGD and Lf competitively reduced the cellular uptake of RGD-Lf-LP, in particular, free RGD played a main inhibitory effect on cellular uptake of RGD-Lf-LP in U87 MG cells, yet free Lf played a main inhibitory effect on cellular uptake of RGD-Lf-LP in hCMEC/D3 cells. RGD-Lf-LP can also significantly increase penetration of U87 MG tumor spheroids, and RGD modification plays a dominating role on promoting the penetration of U87 MG tumor spheroids. The results of in vitro BBB model were shown that RGD-Lf-LPC6 obviously increased the transport of hCMEC/D3 cell monolayers, and Lf modification plays a dominating role on increasing the transport of hCMEC/D3 cell monolayers. In vivo imaging proved that RGD-Lf-LP shows stronger targeting effects for brain orthotopic gliomas than that of RGD-LP and Lf-LP. The result of tissue distribution confirmed that RGD-LF-LP-DTX could significantly increase brain targeting after intravenous injection. Furthermore, RGD-LF-LP-DTX (a dose of 5 mg kg−1 DTX) could significantly prolong the survival time of orthotopic glioma-bearing mice. In summary, RGD and LF dual modification are good combination for brain targeting delivery, RGD-Lf-LP-DTX could enhance brain targeting effects, and is thus a promising chemotherapeutic drug delivery system for treatment of glioma.

6. 한글 초록 요약본:

교모세포종 (GBM) 치료에서 BBB는 항암제의 뇌 전달을 제한하는 주요 장벽임. 본 연구에서는 BBB의 cerebral microvascular endothelial cell과 glioma cell 모두에서 발현되는 integrin α_vβ₃와 lactoferrin receptor (LfR)를 동시에 표적하기 위해 RGD와 lactoferrin (Lf)을 결합한 dual-targeting liposome (RGD-Lf-Lp)을 제작하고 docetaxel (DTX)을 탑재함. RGD-Lf-Lp는 단일 ligand liposome보다 U87MG glioma cell과 hCMEC/D3 endothelial cell에서 높은 uptake를 보였으며, RGD는 주로 tumor penetration, Lf는 BBB transport에 더 크게 기여함. Orthotopic glioma model에서도 dual-targeting liposome의 뇌종양 축적이 가장 높았고 RGD-Lf-Lp-DTX 투여군에서 가장 우수한 항종양 효과와 생존기간 연장이 나타남. 따라서 BBB crossing과 glioma targeting을 동시에 강화하는 dual-ligand strategy의 가능성을 제시한 연구임.

7. 한글 논문 요약본 

7-1) Introduction: Glioma는 수술 후에도 침윤성 종양세포가 남기 쉽고 BBB 때문에 충분한 농도의 항암제를 종양에 전달하기 어려움. Integrin α_vβ₃ 는 cerebral microvascular endothelial cell과 glioma cell에 과발현하며 RGD peptide가 선택적으로 결합함. LfR 역시 BBB endothelial cell과 glioma cell 모두에 발현되며 Lf를 이용한 receptor-mediated endocytosis를 통해 BBB crossing 및 glioma targeting이 가능함. 저자들은 하나의 ligand만 사용하는 것보다 서로 다른 receptor를 이용하는 dual-targeting이 효과적일 것으로 보고 RGD+Lf가 동시에 표면에 결합된 PEGylated liposome에 DTX를 탑재하여 BBB 통과와 glioma targeting을 동시에 증가시키고자 함.

7-2) Methods

-       Nanocarrier: PEGylated liposome

-       Targeting ligand: RGD -> integrin α_vβ₃ / Lf -> LfR

-       Payload: Docetaxel (DTX)

-       세포: U87MG glioma cell, hCEMC/D3 brain endothelial cell

-       In vitro: cellular uptake, uptake inhibition, U87MG spheroid penetration, cytotoxicity, hCEMC/D3 Transwell BBB permeability

-       In vivo: U87MG orthotopic glioma 및 subcutaneous model

-       비교군: PEG-LP-DTX, RGD-LP-DTX, Lf-LP-DTX, RGD-Lf-LP-DTX

-       평가: IVIS biodistribution, tissue DTX concentration, tumor growth, TUNEL, survival

 Orthotopic model에서는 DTX 5mg/kg을 tail vein으로 3일 간격, 총 4회 투여함.

7-3) Results

 7-3-1) RGD-Lf-LP-DTX의 제조 및 특성: RGD와 Lf가 결합된 DTX-loaded liposome은 약 136nm 크기로 안정적으로 제조되었으며 약 78%의 encapsulation efficiency와 지속적인 DTX release를 보였음. Serum 및 4℃ 보관 조건에서도 비교적 안정함.

7-3-2) RGD-Lf dual modification에 의한 cellular uptake 증가: RGD-Lf-LP는 U87MG와 hCEMC/D3 모두에서 단일 ligand liposome보다 높은 cellular uptake를 나타냄. 경쟁적 inhibition 결과, RGD는 U87MG uptake에, Lf는 hCEMC/D3 uptake에 상대적으로 더 크게 기여함.

 7-3-3) Glioma penetration 및 cytotoxicity 증가: RGD-Lf-LP는 U87MG tumor spheroid에서 가장 높은 penetration을 보였으며, 특히 RGD가 깊은 tumor penetration에 주요 역할을 함. DTX를 탑재한 경우에도 RGD-Lf-LP-DTX가 가장 높은 glioma cytotoxicity를 나타냄.

 7-3-4) BBB permeability 및 brain targeting 증가: In vitro BBB model에서 RGD-Lf-LP가 가장 높은 endothelial transport를 나타냈으며 이 과정에서 Lf가 상대적으로 주요 역할을 함. Orthotopic glioma mouse에서도 dual-targeting liposome의 tumor accumulation과 brain DTX concentration이 가장 높았음.

 7-3-5) In vivo anti-glioma efficacy 증가: RGD-Lf-LP-DTX는 subcutaneous glioma에서 가장 강한 tumor growth inhibition을 보였으며 orthotopic model에서는 median survival을 20일에서 32일로 연장함. 뚜렷한 체중 감소나 주요 조직 독성은 관찰되지 않았음.

⁂ Lf는 BBB crossing에, RGD는 glioma penetration에 상대적으로 크게 기여하며 두 ligand를 결합한 dual-targeting 전략이 DTX의 brain delivery와 anti-glioma efficacy를 향상시킴.  

8.대표 Figure : Fig 1. In vivo brain/ glioma targeting