Nov 13, 2023
Engenharia de microfábricas de oxigênio para retardar a progressão do tumor por meio de microambientes hiperóxicos
Volume de comunicações da natureza
Nature Communications volume 13, Número do artigo: 4495 (2022) Citar este artigo
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Embora a hipóxia promova carcinogênese, agressividade tumoral, metástase e resistência a tratamentos oncológicos, os impactos da hiperóxia nos tumores raramente são explorados porque fornecer um suprimento de oxigênio duradouro in vivo é um grande desafio. Aqui, construímos microfábricas de oxigênio, ou seja, microcápsulas de fotossíntese (PMCs), por encapsulamento de cianobactérias adquiridas e nanopartículas de upconversion em microcápsulas de alginato. Este sistema permite um suprimento de oxigênio duradouro através da conversão de radiação externa em emissões de comprimento de onda vermelho para fotossíntese em cianobactérias. O tratamento com PMC suprime a via NF-kB, a produção de HIF-1α e a proliferação de células cancerígenas. O microambiente hiperóxico criado por um implante PMC in vivo inibe o crescimento e a metástase do hepatocarcinoma e tem efeitos sinérgicos juntamente com o anti-PD-1 no câncer de mama. As fábricas de oxigênio de engenharia oferecem potencial para estudos de biologia tumoral em microambientes hiperóxicos e inspiram a exploração de tratamentos oncológicos.
A hipóxia é a característica mais difundida dos microambientes dos tumores sólidos1,2 e decorre de um desequilíbrio entre o suprimento insuficiente de oxigênio e o aumento do consumo de oxigênio pelas células cancerígenas de rápida proliferação. Consequentemente, as células cancerígenas recorrem a múltiplas vias adaptativas e alterações genômicas para sobreviver em ambientes hipóxicos3. O fator de transcrição 1α induzível por hipóxia (HIF-1α), o mediador mais reconhecido de respostas hipóxicas, desempenha um papel central na estimulação da neovascularização em tumores para aumentar o suprimento de oxigênio e nutrientes4. Paradoxalmente, esses vasos são frequentemente organizados de forma irregular (por exemplo, estruturas retorcidas, hiperpermeáveis e com extremidades cegas) e apresentam defeitos na difusão ou perfusão de oxigênio5, resultando em expansões de regiões hipóxicas nos tumores. Concomitantemente, foi relatado que o microambiente hipóxico, uma característica dos tumores malignos, não é apenas a barreira primária que protege o tumor de várias terapias, criando um ambiente de imunossupressão6, ativando a via de reparo do DNA7 e permitindo o fluxo autofágico8, mas também um promotor da carcinogênese9 , invasão tumoral e metástase1,2. Essas descobertas inspiraram a exploração de tecnologias para converter microambientes hipóxicos em microambientes hiperóxicos para estudos de biologia ou terapia de tumores.
É um grande desafio construir um microambiente hiperóxico duradouro em tumores devido à falta de fontes de oxigênio constantes e biocompatíveis. Considerando que os micróbios de algas são os principais fornecedores de O2 na Terra, a fotossíntese em cloroplastos de algas poderia ser potencialmente explorada para suplementos de O2 em tumores. A maquinaria fotossintética requer uma fonte de luz correspondente que emita fótons de 650 a 700 nm. Como as nanopartículas de conversão ascendente baseadas em terras raras (UCNPs) mostraram uma capacidade extraordinária de converter lasers biotransparentes de infravermelho próximo (NIR) em luz visível10, esses materiais podem ser explorados para fornecer fótons disponíveis na fotossíntese. Portanto, levantamos a hipótese de que um microambiente hiperóxico duradouro poderia ser criado pela construção racional de micróbios de algas e UCNPs.
Neste estudo, somos pioneiros em uma microcápsula de fotossíntese (PMC) encapsulando cianobactérias e UCNPs em microcápsulas de alginato (MCs) que podem ser fabricadas por uma técnica de gotícula eletrostática. Quatro cepas de cianobactérias foram submetidas à seleção de aclimatação para adquirir uma cepa adequada para a acomodação de condições fisiológicas. Exploramos de forma abrangente os impactos da radiação NIR, população de células e dose de UCNP na produção de O2 para projetar uma fórmula otimizada de PMCs. Os impactos dos microambientes hiperóxicos criados por PMCs são examinados em nove linhagens celulares de câncer e dois modelos de tumor, incluindo câncer de mama ortotópico em camundongos e hepatocarcinoma transplantado em coelhos.
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>
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