과산소 미세 환경을 통해 종양 진행을 늦추는 미세 산소 공장 엔지니어링

Nature Communications 13권, 기사 번호: 4495(2022) 이 기사 인용

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저산소증은 발암, 종양의 공격성, 전이 및 종양학적 치료에 대한 저항성을 촉진하지만 생체 내에서 장기간 산소 공급을 제공하는 것이 주요 과제이기 때문에 종양에 대한 과산소증의 영향은 거의 연구되지 않았습니다. 본 연구에서는 획득된 시아노박테리아와 상향전환 나노입자를 알지네이트 마이크로캡슐에 캡슐화하여 미세 산소 공장, 즉 광합성 마이크로캡슐(PMC)을 구축합니다. 이 시스템은 남조류의 광합성을 위해 외부 방사선을 적색 파장 방출로 변환하여 오래 지속되는 산소 공급을 가능하게 합니다. PMC 치료는 NF-kB 경로, HIF-1α 생성 및 암세포 증식을 억제합니다. 생체 내 PMC 임플란트에 의해 생성된 고산소 미세환경은 간암종의 성장과 전이를 억제하고 유방암에서 항PD-1과 함께 시너지 효과를 나타냅니다. 엔지니어링 산소 공장은 고산소 미세 환경에서 종양 생물학 연구에 대한 잠재력을 제공하고 종양학적 치료법의 탐구에 영감을 줍니다.

저산소증은 고형 종양 미세 환경의 가장 널리 퍼져 있는 특징이며1,2 암세포의 급속한 증식으로 인한 산소 공급 부족과 산소 소비 증가 사이의 불균형으로 인해 발생합니다. 결과적으로, 암세포는 저산소 환경에서 생존을 위해 다중 적응 경로와 게놈 변화에 의존합니다3. 저산소증 반응의 가장 잘 알려진 중재자인 전사 인자 저산소증 유발 인자 1α(HIF-1α)는 종양의 혈관신생을 자극하여 산소와 영양분 공급을 향상시키는 데 중심적인 역할을 합니다4. 역설적이게도 이러한 혈관은 종종 불규칙하게 조직되어 있으며(예: 뒤틀리고, 과투과성이 있고 끝이 막힌 구조) 산소 확산 또는 관류에 결함이 있어 종양의 저산소 영역이 확장됩니다. 동시에, 악성 종양의 특징인 저산소 미세환경은 면역억제 환경을 조성하고, DNA 복구 경로를 활성화하고, 자가포식 플럭스를 활성화함으로써 다양한 치료로부터 종양을 보호하는 일차 장벽일 뿐만 아니라 발암 촉진제인 것으로 보고되었습니다9 , 종양 침습성 및 전이1,2. 이러한 발견은 종양 생물학 또는 치료 연구를 위해 저산소 미세환경을 고산소 미세환경으로 전환하는 기술 탐구에 영감을 주었습니다.

일정하고 생체에 적합한 산소 공급원이 부족하기 때문에 종양에서 오래 지속되는 과산소 미세 환경을 구축하는 것은 주요 과제입니다. 조류 미생물이 지구상의 O2의 주요 공급원이라는 점을 고려하면, 조류 엽록체의 광합성은 잠재적으로 종양의 O2 보충제를 탐색할 수 있습니다. 광합성 기계에는 650~700 nm 광자를 방출하는 일치하는 광원이 필요합니다. 희토류 기반 상향변환 나노입자(UCNP)는 생체투명 근적외선(NIR) 레이저를 가시광선10으로 변환하는 놀라운 능력을 보여주었기 때문에 이러한 물질은 광합성에서 이용 가능한 광자를 제공하는 데 활용될 수 있습니다. 따라서 우리는 조류 미생물과 UCNP의 합리적인 구성을 통해 오래 지속되는 고산소 미세 환경이 생성될 수 있다는 가설을 세웠습니다.

본 연구에서는 정전기적 액적 기술로 제작할 수 있는 알기네이트 마이크로캡슐(MC)에 시아노박테리아와 UCNP를 캡슐화하여 광합성 마이크로캡슐(PMC)을 개척했습니다. 4개의 시아노박테리아 균주는 생리적 조건의 수용에 적합한 균주를 획득하기 위해 적응 선택을 거쳤습니다. 우리는 NIR 방사선, 세포 집단 및 UCNP 선량이 O2 생산에 미치는 영향을 포괄적으로 조사하여 최적화된 PMC 공식을 설계합니다. PMC에 의해 생성된 고산소 미세 환경의 영향은 생쥐의 정위 유방암과 토끼의 이식된 간암종을 포함하여 9개의 암 세포주와 2개의 종양 모델에서 검사됩니다.

32 °C, S. sp. 6803 and S. elongate. 7942 were able to acclimate to the temperature increments. Stepwise changes from the BG11 medium to DMEM allowed us to acquire an evolved S. sp. 6803 (e-S. sp. 6803) strain, which maintained its activity at 37 °C in DMEM (Supplementary Fig. 3). This strain was therefore selected for PMC construction. We then synthesized a series of UCNPs with different emissions by the crystal growth method10. An Er3+- and Yb3+-doped NaYF4 nanorod (15.3 × 30.2 nm) was found to emit strong fluorescence at 660 nm, perfectly matching the absorbance of chlorophyll α (Supplementary Fig. 4 and Supplementary Fig. 5), which is the prominent component responsible for photosynthesis. Next, we engineered the PMCs by encapsulating algal microbes and UCNPs in the alginate-calcium microspheres under an electrostatic field via an electrostatic droplet generation system. As shown in Fig. 1, the whole process involves four steps: (i) homogenous mixing of algal microbes with UCNPs in alginate sodium; (ii) dispersing of alginate sodium solution into uniform droplets under an electrostatic field; (iii) encapsulating UCNPs and microbes by cross-linked alginate-calcium in CaCl2 solutions; and (iv) coating of alginate-calcium microspheres by poly-L-lysine (PLL), a water-soluble polycation that is resistant to enzymatic degradation and capable of preventing microbe leakage (Supplementary Fig. 6). The resulting PMCs were examined by upconversion luminescence microscopy. While empty MCs and alga-encapsulated MCs had no fluorescence signals, PMCs emitted strong red fluorescence under 980 nm excitation (Fig. 2a). These results indicated that the encapsulated UCNPs thoroughly maintained their optical properties. We comprehensively examined the impacts of microbe density and UCNP concentration on the photosynthetic activity of PMCs (Fig. 2b). An optimized formula of PMCs (3 × 103 algal cells and 0.67 μg of UCNPs per MC) was acquired for efficient oxygen production (1.6 μg/min). The oxygen generation of designated PMCs was dependent on the intensity and exposure time of NIR radiation, indicating a controllable oxygen supply (Supplementary Fig. 7). The encapsulated algal microbes in PMCs survived in DMEM for over 1 month (Supplementary Fig. 8)./p>140 days and were almost cured, as there were no detectable tumour nodules in the livers and lungs by CT imaging and ex vivo examination (Supplementary Fig. 23). To our surprise, luminescent PMCs could still be observed in the liver and maintained spherical morphologies (Supplementary Fig. 23). These results indicated that the hyperoxic microenvironment created by NIR-PMCs could greatly slow tumour progression, inhibit tumour metastasis and enhance the survival rates of hepatocarcinoma-bearing rabbits./p>140 days) than the untreated animals did (average survival time ~27 days) and had no detectable tumour nodes. However, these findings may need more validation across different tumour models./p>Stage II) to establish local control and palliation. The PMCs were deliberately designed to accommodate the interventional device. Although intratumor injection was selected for the administration of PMCs in animals, PMCs could be applied in human patients by transcatheter arterial chemoembolization. To facilitate future clinical applications, the dose and duration time of NIR radiation should be carefully examined. Taking hepatocarcinoma as an example, 900 mW/cm2 NIR radiation at 980 nm was applied to rabbits for 60 min/day. Given that the depth of hepatocarcinoma in rabbits is 0.3–0.7 cm, the tumour received 300–500 mW/cm2 radiation after penetration of the animal belly60. To acquire such excitation intensity in human patients, the radiation dose has to be increased to 2000 mW/cm2 or the duration time should be extended to 180 min because hepatocarcinoma in human patients are often deeper than they are in rabbits61,62,63 and because the intensity of NIR radiation at 980 nm would decline to <30% after penetration of 0.7–1.0 cm belly tissues in humans. However, this amount of NIR radiation may induce hyperthermia damage. Alternatively, NIR-II radiation at 1200–1700 nm would be more suitable for clinical applications, as NIR-II photons have a much deeper penetration capability than NIR lasers at 980 nm64. UCNPs with excitations in the NIR-II region could be exploited to construct PMCs for potential applications in clinics. Since interventional therapy is a conventional treatment in hepatocarcinoma patients, PMCs are a promising implant for clinical applications./p>85% cell proliferation, we increased the culture temperature (1 °C). Otherwise, we sustained the temperature for another 24 h. After 30 days culture, the evolved algal microbes were acquired and preserved in BG11 media at 37 °C for subsequent tests. Next, we repeated the above procedure by stepwise changing medium composition from BG11 to DMEM. The evolved Synechocystis sp. 6803 cultured at 37 °C in DMEM was denoted as e-S. sp. 6803./p>50 μm./p>2 cm for mice or the tumour volume is >80 cm3 for rabbits; (ii) the eating, drinking or movement of animals is severely affected. To develop the hepatic VX2 tumours in rabbits, VX2 cell suspensions (2 × 106 cells, 200 μL) were implanted into the thigh muscles of donor rabbits. Once the tumour sizes were >2 cm (~2 weeks), the donor rabbits were anesthetized by intravenous injection at a lethal dose 2 mL/Kg of xylazine hydrochloride for the harvest of tumour tissues. Each tumour was minced into 1 mm3 piece by ophthalmic scissors under sterile conditions. The recipient rabbits were anesthetized by intramuscular injection of xylazine hydrochloride (250 μL/Kg). A minced tissue fragment was directly delivered percutaneously into the subcapsular parenchyma of the left hepatic lobe of the recipient rabbit by percutaneous puncture technique under a 16-slice CT spiral scan (Brilliance-16, Phillips, USA) guidance. The rabbits were housed and examined by CT imaging until the tumour volumes reached around 1 cm3. The hepatocarcinoma-bearing rabbits with similar tumour size were divided into two groups by throwing dice, including vehicle control (n = 13), NIR-PMC group (n = 13). The rabbits were anesthetized for a single intratumorally injection of PMC suspensions (500 µL, 3.6 × 104/mL) at 14 days. NIR radiations at 900 mW/cm2 were exposed to animals for three intervals (20 min in each interval) each day. The tumour size was monitored by CT scanning (MHCT brilliance 16, Philips, Holland) every 2 weeks./p> 1 indicates antagonism, CI = 1 indicates additivity, and CI < 1 indicates synergy. The tumours were imaged by IVIS imaging spectrum system (PerkinElmer, ME, USA) and Canon camera (Japan). The mice were fully anesthetized by an overdose of sodium pentobarbital (400 mg/kg) and sacrificed to collect tumours and lungs. The tissue samples were stored in liquid nitrogen for cytokine and adenosine measurements or fixed for H&E staining or immunostaining of A2AR, CD4, CD39, CD206 and CD73 expression./p>