Advanced nanomaterials for hypoxia tumor therapy: challenges and solutions

Aravindkumar Sundaram abc, Ling Peng b, Luxiao Chai b, Zhongjian Xie *d, Joice Sophia Ponraj *ce, Xiangjiang Wang a, Guiqing Wang *a, Bin Zhang b, Guohui Nie b, Ni Xie *b, Manavalan Rajesh Kumar f and Han Zhang *b
aDepartment of Orthopaedic Surgery, the Sixth Affiliated Hospital of Guangzhou Medical University, 511508 Qingyuan, Guangdong, China. E-mail: 1918635344@qq.com
bKey Laboratory of Optoelectronic Devices and Systems of Ministry of Education and Guangdong Province, Institute of Microscale Optoelectronics, and Otolaryngology Department and Biobank of the First Affiliated Hospital, Shenzhen Second People's Hospital, Health Science Center, Shenzhen University, 518060 Shenzhen, China. E-mail: xn100@szu.edu.cn; hzhang@szu.edu.cn
cCentre for Advanced Materials, Integrated-Inter-Department of LiWET Communications, Aaivalayam-Dynamic Integrated Research Academy and Corporations (A-DIRAC), 641046 Coimbatore, India. E-mail: joice.ponraj@aaivalayam.com
dShenzhen International Institute for Biomedical Research, 518116 Shenzhen, Guangdong, China. E-mail: zjxie2011@163.com
eDepartment of Micro and Nanofabrication, International Iberian Nanotechnology Laboratory (INL), 4715-330 Braga, Portugal
fInstitute of Natural Science and Mathematics, Ural Federal University, 620002 Yekaterinburg, Russia

Received 31st August 2020 , Accepted 7th October 2020

First published on 15th October 2020


In recent years, nanomaterials and nanotechnology have emerged as vital factors in the medical field with a unique contribution to cancer medicine. Given the increasing number of cancer patients, it is necessarily required to develop innovative strategies and therapeutic modalities to tackle hypoxia, which forms a hallmark and great barrier in treating solid tumors. The present review details the challenges in nanotechnology-based hypoxia, targeting the strategies and solutions for better therapeutic performances. The interaction between hypoxia and tumor is firstly introduced. Then, we review the recently developed engineered nanomaterials towards multimodal hypoxia tumor therapies, including chemotherapy, radiotherapy, and sonodynamic treatment. In the next part, we summarize the nanotechnology-based strategies for overcoming hypoxia problems. Finally, current challenges and future directions are proposed for successfully overcoming the hypoxia tumor problems.


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Aravindkumar Sundaram

Aravindkumar Sundaram obtained his B.E. in Biomedical Engineering (Sri Ramakrishna Engineering College, Coimbatore, India) and M.Tech in Nanoscience and Technology (Regional Campus of Anna University Coimbatore – India) in 2015 and 2018, respectively. After graduation, he joined as an Assistant Professor at the Salem College of Engineering and Technology, India. He is now an early stage research fellow at the Centre for Advanced Materials Aaivalayam-DIRAC, Coimbatore, India. His research interest focuses on the design and development of two-dimensional nanomaterials for biomedical applications including cancer therapeutics and diagnostics.

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Zhongjian Xie

Dr Zhongjian Xie received his PhD from University of Lyon, France, in 2016. Then, he joined Prof. Han Zhang's lab in Shenzhen University as a post-doc. Now, he is a full professor at the Shenzhen International Institute for Biomedical Research (SIIBR). His research interests focus on the application of two-dimensional materials in biological and environmental applications.

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Joice Sophia Ponraj

Dr Joice Sophia Ponraj is the Director-Research Professor at CAM, Aaivalayam-DIRAC, India and a Research Fellow at INL, Portugal. Joice is a recipient of the DST INSPIRE Faculty Award and Marie-Curie CoFund. Her research interests include the synthesis and study of 2D materials and semiconducting nanostructures for bionanophotonics, 2D-based cancer therapeutics, micro/nano fabrication, graphene-2D wearable electronics, and optoelectronics. Joice received her PhD from Anna University, India. She was awarded three times with ICTP-TRIL to work in IMEM-CNR, Parma, Italy, “Italian-Indian Bilateral Programme” hosted by University of Ferrara, Italy and AICTE-NDF, India during her PhD. She did post-doc at Suzhou University, China and served as an Assistant Professor in UIST, North Macedonia and Bharathiar University, India.

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Han Zhang

Dr Han Zhang received his B.S. degree from Wuhan University (2006) and received a PhD from Nanyang Technological University (2010). In 2012, he joined the College of Optoelectronic Engineering (Collaborative Innovation Centre for Optoelectronic Science Technology) at Shenzhen University in Shenzhen as a full professor. His current research is on ultrafast, non-linear photonics, and biomedicines of 2D materials.


1. Introduction

Since the first discovery of cancer by Hippocrates (460–370 BC),1 it has become the main causative factor of death across the world and a major threat to public health.2 The current therapeutic strategies for cancer treatment include surgery,3 chemotherapy,4 and radiation therapy (RT).5 However, their unavoidable side effects such as immune system damage, destruction of healthy cells, cardiotoxicity, and arrhythmia remain unaddressed.6–8 Therefore, the complete eradication of tumors without side effects requires efficient modification of traditional methods and development of novel therapeutic modalities.9,10 With the assistance of nanotechnology and nanomaterials, multimodal hypoxia tumor therapies including photodynamic therapy (PDT),11 immunotherapy (IMT),12 targeted therapy (TT),13 photothermal therapy (PTT),14 and sonodynamic therapy (SDT)15 are extensively explored for treating hypoxia tumor.

Nanotechnology is one of the main upcoming technologies in the field of science, which builds nanomaterials by manipulating matter or atoms at the microscopic or subatomic level.16 Engineered by nanotechnology, nanomaterials have superior properties such as (1) a tiny scale to allow them to cross the cellular barrier, (2) a large active surface area to enhance the drug loading capacity, and (3) quantum confinement with superior optical, magnetic, and electrical properties.17–20 Thus, nanomaterials can afford a variety of opportunities for cancer-related therapeutic, diagnostic, and theranostic applications.21–24 For example, they can target their action through functionalization with small molecules, uptake through the enhanced permeability and retention (EPR) effect, prolong the circulation time of drugs, and protect drugs from rapid degradation.25

The most commonly used nanomaterials in cancer therapeutics are chitosan,26 polylactic-co-glycolic acid,27 gold nanoparticles,28 silver nanoparticles,29 silica nanoparticles,30 iron oxide nanoparticles,31 and two-dimensional nanostructures (black phosphorous32,33 and others34,35). These materials are exploited as (1) carriers for drugs in chemotherapy,36 (2) sensitizer cargo for RT,37 PTT,38 PDT,39 and SDT,40 (3) imaging agents of computerized tomography (CT), magnetic resonance imaging (MRI), and photoacoustic imaging (PAI),22,41 and (4) sensing materials in the electrochemical and electroluminescence sense.42,43 However, limited previous reports are available to describe nanoparticle-mediated hypoxia overcoming the tactics and tricks. Giving more details for exploring this research area is the main purpose of this review. On this basis, we have organized the review by focusing on the development of advanced nanomaterial-based therapeutic approaches, especially from the angles of radiotherapy, chemotherapy, photodynamic therapy, sonodynamic therapy, and immunotherapy as well as recent strategies such as HIF-1 delivery and gene silencing, which are relatively new and not yet discussed in detail, as shown in Fig. 1. Furthermore, we have summarized the role of nanomaterials and the consequent therapeutic approaches in treating hypoxia tumor44 by explaining how hypoxia can be a potential problem for the successful treatment of cancer and which advanced nanomaterials and nanotechnological based tactics can be used to overcome it. Finally, the article concludes with a critical assessment of the potential of the nanotechnological solutions, which often present severe drawbacks due to economic, upscaling, reproducibility, or safety reasons.


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Fig. 1 Schematic representation of review organization.

2. Origin and relation between hypoxia and tumor

Hypoxia refers to the phenomenon of diminished oxygen levels (pO2 < 10–15 mmHg) in the tumor cell and the tumor microenvironment (TME).45,46 It appears significantly in massive tumors and has been evidenced by many experiments. In the view of chemistry, the reduction of O2 electron transport47 in TME arises from the inequality of O2 diffusion/perfusion in distorted blood microcapillaries and the vessels of primary avascular tumors.48 Diffusion-limited hypoxia is generally called as chronic hypoxic and results from the rise in hypoxic cells in zones nearby the necrotic regions (>70 μm distant from the blood capillaries). Typically, these cells die within 4 to 10 days. Perfusion-limited hypoxia, also called acute or fluctuating hypoxia, ensues from the development of hypoxic cells on a small scale downstream of the perfusion-impaired vessels. Perfusion-limited hypoxia can be temporary or recurring and results in the overexpression of vital transcription factors of hypoxia-inducible factor (HIF)-heterodimeric alpha or beta in regular and malignant cells, leading to the creation of therapeutic resistance.49

Thus far, most of the therapeutic strategies, including chemotherapy, RT, PDT, SDT, and IMT, depend primarily on controlling the oxygen concentration in TME. The clear evidence of increased drug-resistance under hypoxia is obtained by using etoposide and doxorubicin (DOX), in particular, in human and non-human cells. The study revealed that the instantaneous increase in the drug-resistance in human glioma cells arises from the alteration of their phenotypes.50–54 In the RT, PDT, and SDT modalities, oxygen is the primary source of reactive oxygen species (ROS)/free radical generation in cancer cell necrosis. In particular, insufficient oxygen supply leads to a lower production of singlet oxygen and a higher accumulation of glutathione (GSH) in TME. Thus, the recovery of oxygen concentration in TME is essential for cancer treatment in RT, PDT, and SDT modalities. By IMT, hypoxia causes the over-production of adenosine, which is a T-cell suppressor and acts as a critical factor for activating immune action against the cancer cells.55,56 In particular, hypoxia causes enzyme activity reduction of prolyl hydroxylase domain 2 (PHD2), which is against the prevention of HIF-1α hydroxylation and the promotion of hypoxia-inducible gene transcription.57 Subsequently, the promotion of tumor growth and malignant phenotypes though these HIF-1 genes by the sequential reactions of angiogenesis, glucose metabolism, extracellular matrix (ECM) remodeling, epithelial-to-mesenchymal transition, cell survival, proliferation, and endogenous production of nitric oxide (NO) is limited.58,59

The visualization and quantification of hypoxia can be done with imaging contrast agents (PET, MRS, MRI, NIRS, and EPR) and tracers. Owing to its non-invasive nature, this technique lacks sufficient spatial resolution to accurately detect patterns of hypoxia within the TME.60,61 Otherwise, microelectrodes such as Eppendorf oxygen probes are utilized to profile extracellular pO2 and pH levels,62,63 while providing a quantitative analysis of the oxygen levels. However, they are technically demanding to use and also impart poor spatial resolution. A common method for quantifying hypoxia in stained histological sections is binary thresholding, in which the fraction of pixels above a predefined threshold is measured to show the hypoxia-positive area as a percentage of tumor area.64,65 Since hypoxia is a continuous gradient within the tissues, there is no universally accepted threshold for discriminating hypoxia from normoxia.66 Several studies have employed different methods for measuring hypoxia gradients relative to blood vessels. One popular method is vessel distance analysis (VDA), particularly due to the development of image analysis platforms that are capable of this type of analysis. VDA computes the mean marker intensity in an image object [pixel, segmented cell, or some other region of interest (ROI)] as a function of the distance to a vessel.

In short, the degree and duration of hypoxia presence determine the effect of tumor therapeutics. For example, it may promote the growth and metastasis of cancer cells or cause cell death.67 Severe and prolonged hypoxia can induce cell death through multiple proteomic changes such as cell-cycle arrest, differentiation, apoptosis, and necrosis. Alternatively, proteomic changes in the cells can also be induced under moderate and short hypoxic conditions for the cell to survive.68–70 Hence, there exists a necessity for overcoming hypoxia in cancer therapeutics.

3. Development of nanomaterial-mediated hypoxia target cancer therapy

Advanced nanotechnological tools allow researchers to ingrain and develop novel and advanced nanostructures with superior physiochemical properties. In particular, their sizes and shapes open up the potential for crossing various biological barriers under hypoxic conditions. The tiny size allows for easy access into the cell and various cellular compartments including the nucleus. A multitude of substances are currently being studied for the preparation of nanoparticles applied in drug delivery, varying from biological substances such as albumin, gelatin, and phospholipids for liposomes to more chemical substances including polymers and solid metal-containing nanoparticles. It is obvious that the potential interaction with tissues and cells together with the potential toxicity greatly depend on the actual composition of the nanoparticles. Furthermore, the chemical properties (pH and surface charge), and magnetic and photonic properties compliment the ability of nanomaterials to effectively release the drug, oxygen carriers, and other agents in a hypoxic environment, which are not usually easily accessible.

3.1. Nanomaterial-mediated radiation therapy of hypoxic tumors

Nowadays, radiotherapy (RT) has become the most common and vastly applied therapeutic strategy (>50%) in cancer treatment.71 Through external or internal irradiation with highly energetic X-rays and gamma (γ) rays, DNA destruction and development of oxidative stress along with free radicals are induced to kill the tumor cells.72 In the earlier days of RT, nano-sized elements such as gold73 and rare earth elements74 were used for enhancing the sensitivity of tumor cells towards radiation and accelerating (ROS) generation, thus inducing cellular cancer damage.75,76 At this junction, the lack of oxygen concentration (hypoxia) impedes the radiation sensitivity and the formation of ROS.77 In addition, the presence of reductant chemical groups (–SH) under hypoxia78 could cause reduced oxidation and increased reduction functionalities of radiation sensitizers, which leads to radiation insensitivity in tumor cells and the failure of RT.

To overcome hypoxia resistance, oxygen modulation,79 high atomic number (Z) element nanoparticles,80 and advanced multifunctional nanomaterials, i.e., catalytic nano-enzymes are introduced to RT.81 These approaches make a difference through (1) the increase in radiation energy within the tumor cells to enhance radiation-induced DNA damage, (2) to enable the free exchange of substrate H2O2 and product O2 to maintain a high catalytic activity/stability, and (3) to effectively deliver the radiosensitizer/anti-cancer drug into the tumor by the EPR effect, as graphically depicted in Fig. 2.82


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Fig. 2 Schematic illustration of various developments in RT-based hypoxia tumor therapy. Reproduced (adapted) with permission.82 Copyright 2016, Dove Medical Press.
3.1.1. Radio-sensitizers. Radiosensitizers are substances that are built up by single or group of elements intended to sensitize the tumor cells for radiation. They promote the formation and fixation of ROS along with the oxidative stress at molecular levels so as to impel cell death by subsequently preventing the cellular repair mechanism. Some nitroaromatic and heterocyclic compounds are known to act as hypoxic cell radiosensitizers, mimicking oxygen molecules with enhanced electron-affinity radiation sensitizing properties.83 The most common and conventional hypoxic cell radiosensitizers are nicotinamide, metronidazole, and their analogs (misonidazole, etanidazole, nimorazole). Generally, these compounds are lipophilic and cannot be delivered orally since they can intrude into the nervous system to elicit neuropathy and other side effects. Hence, they were encapsulated into biocompatible and non-toxic nanomaterials, including iron oxide,84 gold nanoparticles,85 and PLGA nanoparticles.86 In addition, their physio-chemical and pharmacokinetic properties enhance the sensitivity towards the tumor sites for overcoming hypoxia-induced therapeutic radio resistance.87–89 For instance, misonidazole was incorporated into the pH-responsive liposomal superparamagnetic iron oxide-nanoformulations.84 This system displayed a higher entrapment efficiency (30%) in the hypoxic region with few limitations that are needed to account for their therapeutic performance lags. Similarly, metronidazoles were encapsulated in gold nanoparticles together with a chemotherapeutic drug (DOX). The study revealed the enhanced radiation sensitivity of the tumor cells by angiopep-2 modification, which was used to target the cells.85 Importantly, many of these metronidazoles were abandoned in phase III clinical trials. Alternatively, anti-cancer drugs (e.g., paclitaxel and tirapazamine (TPZ)) were also utilized as radiosensitizers for RT because they could precisely arrest the G2/M phase in the cell cycle by interacting with mitotic spindle function.90 As an illustration, paclitaxel was encapsulated into poly(D,L-lactide-co-glycolide) (PLGA) nanoparticles with a dimension from 200 to 500 nm by oil in water (o/w) emulsification-solvent evaporation method.86 In this illustration, the increased blockage of the G2/M phase along with the improved radio-sensitization effect on the hypoxic HeLa and HepG2 cells was observed.90 These studies indicate that nanoformulation-based conventional hypoxic radiosensitizers possess satisfactory performance in RT enhancement.
3.1.2. High atomic number element nanomaterials. The high atomic number (Z) element nanomaterials, e.g., gold,91 silver,92 and gadolinium,93 have been selected as effective radiosensitizers as well as chemotherapeutic agents in combination with RT. Their interesting physico-chemical and pharmacokinetic properties and tunable nanostructures make them favorable agents for RT. For instance, high Z number elements can trigger the nanoparticles to undergo several physical and chemical reactions, such as the Compton effect, photoelectric effect, and Auger effect, to initiate ROS formation for the destruction of cancer cells.

Among various metal-based nanostructures, gold nanoparticles have received colossal attention due to their excellent properties such as (1) excellent biocompatibility, (2) chemical stability, (3) easy surface modification, and (4) high X-ray absorption coefficients.94 A spectrum of studies has revealed that the biological mechanisms of radio-sensitization of gold nanoparticles might be involved in cell cycle interruption and oxidative stress-mediated apoptosis, necrosis, or DNA damage.95–97 Besides, Monte Carlo calculations predicted the effectiveness of gold nanoparticles as potential radiation sensitizers in multiple cancer cell lines and radiation sources.94 Initially, Lei Cui et al. reported the tremendous radio-sensitization effects of gold nanoparticles under normoxia, acute hypoxia, and chronic hypoxia conditions, and their inhibition of the DNA restoration mechanism along with cell cycle synchronization after irradiation.98 Interestingly, gold nanoparticles showed enhanced cell surviving fraction (SF) ratio of 0.22 ± 0.08 in chronic hypoxia with established inhibition in the homologous recombination repair pathway. Alternatively, a higher radio-sensitization effect was observed with 1.9 nm gold nanoparticles under moderate hypoxia (1% O2, DEF = 1.39) conditions.42 However, automatic DNA restoration under hypoxia remains a challenging task. To explore the DNA repair mechanism in hypoxia, Xuan Yi et al. developed a gold-incorporated MnO2 core–shell nanostructure.91 In this system, the gold core acted as a radiosensitizer for interacting the with X-rays and the MnO2 shell catalyzed the conversion of H2O2 into O2 in the tumor cells. The result revealed that with the increase in oxygen concentration, it could decrease the thiol reaction between the tumor and the damaged DNA, and enhance the RT outcomes. Similarly, gold nanoparticles were embedded on the SiO2 core surface to evaluate their anti-tumor efficacy in CT26 colon cancer.99 Their preparation and radiation–sensitization effects are depicted in Fig. 3. In vitro analysis showed the composite system has an improved radiosensitivity and decreased tumor cell viability compared to individual RT. However, the system's sparse vasculature networks under hypoxia reduced the targeting availability of the gold nanoparticles towards the tumor cells. For this, alive attenuated salmonella bacteria-based vehicles with gold nanoparticle (AuNP) decoration were developed.100 The system evidenced a higher accumulation of gold nanoparticles in the center of the tumor. In particular, the tropism of the bacteria caused the delivery of AuNPs to the most radioresistant part of the tumors. Recently, platinum nanoparticles were also employed as radiosensitizers in hypoxia tumor therapy.101


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Fig. 3 Nanomaterials for the radiotherapy of hypoxic tumors. (A) Schematic representation of nanoparticle preparation; (B) MTT viability assay; (C) apoptosis assay. Reproduced (adapted) with permission.99 Copyright 2016, The Korean Society for Radiation Oncology.

Compared to gold nanoparticles, silver nanoparticles (AgNPs) were reported as more suitable radiosensitizers in terms of the higher cost-effectiveness with excellent properties. For instance, Zhujun Liu et al. performed the radiosensitizing evaluation of silver nanoparticles in hypoxic glioma cells and observed satisfactory IC50 value with an enhanced sensitization enhancement ratio (SER) of 1.78.92 In addition, promoted apoptosis and improved autophagy were found to contribute to the radiosensitizing ability of silver nanoparticles in hypoxic cells.102

The studies reported that both gold and silver-based nanoparticles showed satisfactory performances in RT. However, either of them have some problems to be solved. In the case of gold nanoparticles, these are (1) long blood half-life, (2) accumulation and renal clearance problems, (3) skin color change, (4) and cost-effectiveness. Similarly, people ingesting silver nanoparticles at high levels permanently turn the skin to blue color. Hence, for future clinical trials, novel high Z number nanoparticles are advised to be developed. For safety issues, more nanostructures (e.g., iodine-based nanoparticles103 that are almost colorless, non-toxic, lower cost, and have reasonable clearance) are encouraged to be created for RT.

Besides, high Z number metal-based nanoparticles, several rare earth elements such as ytterbium104 and gadolinium93 based materials are also identified as radiation sensitizers. However, their inherent toxicity significantly reduces the usage in therapeutic and imaging applications.105 The effectiveness of such materials in hypoxic tumor treatment remains a debate.106 Researchers should pay more attention to radioprotectors in order to avoid the side effects and non-malignant cell death, and further, for better enhancement of RT. Moreover, this strategy has been extended to the design of novel types of nanoformulations with intriguing multi-functionalities for RT enhancement. The nanomaterials used for overcoming hypoxia are summarized in the list given in Table 1. Besides, a proper understanding of the nanoparticles’ role in hypoxia therapy by RT can improve the treatment strategy for higher outcomes in real tumor environments.

Table 1 List of various nanomaterials in RT-based hypoxic tumor therapy
S. No. Nanoformulation Sensitizer Cancer type Strategy Ref.
1 AuNPs (2.7 nm) Gold Breast cancer AuNPs enhance the radio-sensitization effect under hypoxia by inhibiting post-irradiation DNA repair and cell cycle synchronization. 98
2 Tirapazamine-conjugated AuNPs with PEG (16.6 nm) Gold Liver cancer TPZ undergo the reduction reaction by the secondary electrons generated from the AuNPs along with the amplification of OH* radical formation. Further enhances the radiation sensitivity of gold nanoparticles in hypoxic cells. 169
3 Au@MnO2-PEG nanoparticles (100 nm) Gold Breast cancer Au act as a radiosensitizer and MnO2 act as a catalytic enzyme. Gold interacts with X-rays to produce charged particles for improved cancer killing. The MnO2 triggers the decomposition of H2O2 in the tumor microenvironment to generate oxygen and overcomes hypoxia-associated RT resistance. 91
4 Paclitaxel-loaded PLGA (200 to 500 nm) Paclitaxel Liver and cervical cancer Paclitaxel block cells in the G2/M phase and enhances the radiation effect. 86
5 Hydrophilic terpolymer-protein-MnO2 or hydrophobic polymer-lipid-MnO2 (140 and 170 nm) MnO2 Breast cancer The hybrid nanostructure gives biocompatibility and MnO2 nanoparticles overcome hypoxia by the effective conversion of H2O2 into oxygen, which further enhances radiation therapy. 170
6 Albumin-MnO2 (50 nm) Breast cancer MnO2 attenuates hypoxia by the conversion of H2O2 into oxygen for the enhancement of radiation effect. 141
7 Catalase-loaded TaOx nanoshell (127 nm) TaOx Breast cancer Catalase performs the catalytic conversion of H2O2 inside the tumor microenvironment and improves tumor oxygenation for the effective sensitization of TaOx. 147
8 Porous PtNPs (115.6 nm) Pt Lung cancer AuNPs enhances the radiosensitization effect under hypoxia by inhibited post-irradiation DNA repair; it did not lead to cell cycle synchronization. 101


3.2. Nanomaterial-mediated chemotherapy of hypoxic tumors

Surmounting of hypoxia-induced drug resistance in chemotherapy remains a big challenge. The hypoxia-inducible factor 1-alpha (HIF-1α) is a potential transcriptional regulator protein, which will be overexpressed and raised under hypoxic conditions. It acts as a highly proven factor of the establishment of chemotherapy resistance in cancer cells. Under low oxygen conditions, HIF-1α is stabilized and formed as dimers with HIF-1β (another subunit of HIF-1). The generated dimers are subsequently translocated into the nucleus part to regulate the transcription of over 100 target genes. These genes can be implicitly or explicitly involved in drug resistance and cause the failure of chemotherapy. Specifically, Qiong Ma et al. showed that the downregulation of SKA1 gene expression was the main factor for the increase in the chemo-resistance in bone cancer cells.107 Moreover, the mechanism of gene regulation and its biological functions in hypoxia exertion are not well identified. To date, great nanotechnological efforts have been undertaken to overcome hypoxia-induced chemo-resistance, or rather, to understand hypoxia-activated drug delivery principles. For instance, hyaluronic acid (HA)-coated doxorubicin-loaded MnO2 nanoparticles (diameter ∼180 nm) were developed to improve the tumor oxygenation level and to down-regulate the expression of HIF-1α and vascular endothelial growth factor (VEGF).108 The combination of nanoparticles and doxorubicin highlighted the satisfactory increase in the apparent diffusion coefficient (ADC) values (42%) and significantly inhibited the proliferation and growth of breast cells. Interestingly, HA-modified MnO2 NPs could target tumor-associated macrophages (TAM) to release large amounts of O2, which was secreted by H2O2 (Fig. 4).
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Fig. 4 Nanomaterials for the chemotherapy of hypoxic tumors. Effect on the apparent diffusion coefficient (ADC) of the tumors and tumor cell proliferation after treatment with Dox and Man-HA-MnO2 NPs: (A) changes in ADC; (B) increase in the ADC values; (C) cell proliferation images; reproduced (adapted) with permission.108 Copyright 2016, American Chemical Society.

Despite the promising performances of chemotherapy agents, vasculature under hypoxia may cause the failure of chemotherapy. Hence, chemotherapy is always combined with other therapeutic strategies and bio-reductive drugs.109 As a proof, TPZ (a hypoxia active bioreductive anti-cancer drug) and W18O49 were encapsulated in PEG-PCL nanoparticles to evaluate chemo and PTT therapy.110 In this study, the hypoxic environment was artificially created by the absorbance of 800 nm light for effective hypoxia, which is responsible for TPZ release and activation. Then, TPZ was simultaneously activated to induce cytotoxic effects. This system provided new insights into simultaneous hypoxia-activated chemotherapy and photothermal therapy. Although TPZ and AQ4N (1,4-bis[[2-dimethylaminoethyl] amino]-5,8-dihydroxyanthracene-9,10-dione bis-N-oxide dihydrochloride) have been proposed for hypoxia-activated chemotherapy111 and have shown satisfactory performance in treating cancer, their cytocompatibility and reproducibility remains unknown. Moreover, the mechanism of hypoxia intensification and hypoxia-activated chemotherapy is still undecided.

In recent years, chemoradiation therapy induced by the mixed effects of chemical drugs and radiation has attracted more attention in treating hypoxic tumors. For example, radiation-sensitive MnO2 nanoparticles of 140 nm in size contained within albumin and paclitaxel drugs were formed against colorectal cancer.112 The MnO2 shell reacted with H2O2 in an acidic environment and sustainably produced oxygen to overcome hypoxia, thus inhibiting the expression of HIF-1α effectively by modulating various bio-functions including proliferation, angiogenesis, and energy metabolism. Similarly, lipid-based polymeric nanoparticles, loaded with Mis (a hypoxic radiosensitizer)-based poly-prodrug (P-(MIs)n) and DOX,113 indicated cogent accumulation and internalization of nanoparticles in the hypoxic glioma site by the endocytosis process. After that, DOX was rapidly released in the cytoplasm through the conversion of the hydrophobic core into the hydrophilic core, and subsequently moved into the nuclear part of the tumor cells for combined radiosensitization effects and enhanced chemoradiotherapy. Similar results were also observed when using cis-platin and HSA pro-drugs.114

Notedly, the combination of chemotherapeutic agents with other therapeutic modalities is limited in functionality due to less compatibility, low cellular uptake, enzymatic degradation, and lack of targeted and sustained release. Therefore, it is essential to develop novel nano-agents, which have 3 multi-functionalities and improved biocompatibility for RNA/gene delivery systems, for hypoxic tumor therapy. Perche et al. first reported hypoxia-induced siRNA uptake and gene silencing by using a polymeric micellar nanocarrier of 100 nm size115 and obtained enhanced hypoxia-activated in vitro green fluorescent protein (GFP) silencing as compared to the previous 200 nm siRNA formulation.116 Specifically, the polymeric nanocarrier protected the siRNA by avoiding rapid degradation and promoting cellular internalization along with endosomal escape. Moreover, siRNA-based gene silencing has drawn increasing interest in cancer therapeutics with limited drawbacks for clinical translations, including rapid enzymatic degradation and poor cellular uptake.117Table 2 lists the drug and nanoparticle combinations, which have been used so far in hypoxic tumor treatments.

Table 2 List of various nanomaterials in the chemotherapy of hypoxic tumors
S. No. Nanoformulation Sensitizer Cancer type Strategy Ref.
1 DOX and HA-based MnO2 nanoparticles DOX Breast cancer MnO2 modulates hypoxia favorable for DOX by the conversion of H2O2 into O2. 108
2 TPZ loaded-W18O49 nanoparticles TPZ Cervical cancer The W18O49 unit gives rise to a more hypoxic tumor microenvironment and activates the TPZ to achieve hypoxia-activated chemotherapy. 110
3 ALP-(MIs)n/DOX DOX Colon cancer MIs increased the radio-sensitivity of the radio-resistant hypoxic cells, enhancing the DNA damage induced by the ionizing radiation. The released DOX was transported to the nucleus to kill the tumor cells and, when combined with the P-(MIs)n radio-sensitization effects, enhanced the effect of chemoradiotherapy. 171
4 TPZ and PEG-based nanoparticles TPZ Breast cancer PDT induces further hypoxia and angiogenesis favorable for TPZ. Under hypoxia, TPZ enhances the chemotherapy effect. 172
5 DOX-loaded polymeric micelles DOX Breast cancer PDT induces the enchantment of hypoxia and the release of DOX, which further enhances the chemotherapy. 173
6 TPZ@Ce6-PEG complexes TPZ Oral cancer Combinational PDT, PTT, and RT induce hypoxia and favor the enhancement of TPZ activity under hypoxia. 174
7 DOX-loaded Fe3O4, mesoporous SiO2, and Au2O-based nanoparticles DOX Cervical cancer Hypoxia cells are targeted by magnetic iron oxide nanoparticles and gold performs the conversion of H2O2 into O2. Subsequently, DOX reacts with O2 and enhances the chemotherapy. 175


3.3. Nanomaterial-mediated photodynamic therapy of hypoxic tumors

In oxygen-dependent photodynamic therapy, hypoxia seems to be undesirable and decreases the effectiveness of cancer treatment. In general, inadequate exposure to light as well as the overexpression of hypoxia-associated regulatory genes (such as gene encoding P-glycoprotein) and proteins (such as HIF-1α) facilitate the emergence of multi-drug resistance and further impedes PDT.118,119 More specifically, the consumption of oxygen during PDT further potentiates hypoxia and then blocks the cytotoxic effect of the photosensitizers, leading to treatment failure and drug resistance (Fig. 5).120 Therefore, it is crucial to address the hypoxia problem in PDT.
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Fig. 5 Schematic organization of various nanotechnological approaches and mechanisms in PDT. Reproduced (adapted) with permission.120 Copyright 2016, Elsevier.

For instance, MnO2 combined with chlorine e6 (Ce6) photosensitizer nanoparticles was developed to overcome hypoxia resistance and further achieve tumor-specific PDT.121 In this, Ce6 was activated under 661 nm laser irradiation for the subsequent conversion of H2O2 into O2, leading to enhanced therapeutic performance of both in vivo and in vitro models compared to free Ce6. Similarly, Lin et al. formulated MnO2 nanoparticles contained human serum albumin (HSA)122 and used them to obtain two-fold 1O2 generation and 3.5-fold O2 in orthotopic bladder cancer. Irrespective of the successful use of MnO2 nanoparticles in PDT, its unavoidable processes such as poor biocompatibility, difficulties in systemic delivery, transitory O2 generation effects, and continuous external activation, can lead to the failure of the treatment. In the future, researchers should consider these problems for the invention of novel PDT agents. In particular, MnFe2O4 nanoparticles with Ce6 photosensitizer were used as Fenton catalysts for generating singlet oxygen via the Fenton reaction in the H2O2-rich cancer microenvironment.123 Under hypoxic conditions, western blot reported a dramatic decrease in the HIF-1α signal intensity with the increase in nanoparticle concentration. It also suggested the development of continuous O2 generating (MnFe2O4) formulations for alleviating hypoxia; the mechanisms are depicted in Fig. 6. In addition, H2O2-responsive delivery cargos were also developed for the sustained release of O2. For example, catalase and methylene blue (MB) were encapsulated in PLGA nanoparticles. The system achieved the self-sufficiency of O2 and controlled 1O2 release in the PDT process.124 It is essential to emphasize that the PDT efficiency can be improved by higher catalase-activation and cellular targeting of αvβ3 integrin, whereas the toxicity is negligible.


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Fig. 6 Nanomaterials for the PDT of hypoxic tumors. Intracellular oxygen generation and hypoxia alleviation performances: (A) schematic illustration of catalytic in situ oxygen generation; (B) O2 generation in H2O2 solution; (C) repetitive catalytic ability of the nanoparticles; (D) singlet oxygen measurement; (E) western blots of HIF-1α expression in U-87 MG cells; (F) cell viability assay under normoxic conditions; (G) cell viability assay under the hypoxic condition. Reproduced (adapted) with permission.123 Copyright 2017, American Chemical Society.

Although PDT is already an effective modality for treating cancer, it is more recommended to design and select PDT agents with enhanced cytocompatibility and multi-functionalities for combinational therapeutics. The recent nanoformulations of the PDT process used for treating hypoxia tumors are summarized in Table 3.

Table 3 List of nanomaterials in the PDT of hypoxic tumors
S. No. Nanoformulation Sensitizer Cancer type Strategy Ref.
1 MnFe2O4 nanoparticle-anchored mesoporous silica nanoparticles Ce6 Brain cancer MnFe2O4 nanoparticles convert H2O2 into oxygen by the Fenton reaction and relieve hypoxia, enhancing the sensitization effect of Ce6. 123
2 Catalase MB. black hole quencher-3 (BHQ-3) doped PLGA nanoparticle Methylene blue Breast cancer Catalase performs O2 conversion from H2O2 and facilitates the sensitization effect of MB towards the enhancement of PDT. 124
3 Ce6-loaded PEG-modified MnO2 nanoparticle Ce6 Breast cancer MnO2 facilitates the conversion of H2O2 into O2 towards the enhancement of Ce6 sensitization effect for better PDT effect. 121
4 Ce6-loaded, HSA-coated MnO2 nanoparticles Ce6 Bladder cancer MnO2 converts H2O2 into O2 and enhances the sensitization effects of Ce6. 122
5 Perfluorocarbon nanoparticles IR780 Colon and breast cancer PFC delivers oxygen into hypoxic cells and modulates TME favorable for the PDT effect. 137
6 Paclitaxel and Fe(III)-complexed porous coordination network Porphyrins Pancreatic cancer Fe(III) perform a Fenton reaction to convert H2O2 into O2 to enhance the chemo and PDT effects. 176
7 Graphdiyne oxide nanosheet Ce6 Breast cancer Graphdiyne performs the catalase reaction and enhances the PDT effect of Ce6. 177


3.4. Nanomaterial-mediated sonodynamic therapy of hypoxic tumors

Similar to PDT, sonodynamic therapy (SDT) is also a widely accepted therapeutic strategy in cancer treatment. It features enormous gains with cost-effectiveness, safe modality, and deeper penetration depth. This modality is affected by hypoxia due to the following two factors: (1) low oxygen concentration and (2) high concentration of GSH. In other words, a reduced level of oxygen concentration could influence the degree of singlet oxygen generation and a high concentration of GSH causes the rapid consumption of the formed singlet oxygen. Encouragingly, to date, several effective nanotechnological methods are available for outpacing hypoxia resistance in SDT, as represented in Fig. 7. For example, SDT is performed against the pancreatic cancer model (BxPc-3) with the help of oxygen-carrying lipid-stabilized microbubbles (MBs).125 As an SDT sensitizer, rose bengal could activate the microbubbles in the hypoxic condition to deliver oxygen at the tumor site and further generate ROS to kill the cancer cells. The in vivo evaluation of the pancreatic tumor mice model showed a 45% shrinkage in the tumor volume after a five-day treatment, which represented a remarkable therapeutic performance of the microbubbles in the hypoxic environment by SDT. Better efficiency (270-fold higher) was obtained through combination with the antimetabolite gemcitabine (Gem, an anti-cancer drug). For addressing size-related issues and breaking hypoxia resistance, self-oxygen generating nanoparticles were developed for treating pancreatic cancer.126 It has been reported that hypoxia could be overcome by incorporating perfluorocarbon FC-chain into mesoporous organosilica nanoparticles with the IR780 sonosensitizer. Under irradiation, the FC-chain provided sufficient oxygen, leading to ROS generation to diminish the hypoxic-pancreatic cancer cell line (PANC-1 cells). The synthesis of FC chain-based nanoparticles and the strategies for overcoming hypoxia are shown in Fig. 8.
image file: d0nr06271e-f7.tif
Fig. 7 Schematic illustration of the different strategies in the sonodynamic therapy of hypoxic tumors.

image file: d0nr06271e-f8.tif
Fig. 8 Nanomaterials for the sonodynamic therapy of hypoxic tumors. The synthetic process and action mechanisms: (A) schematic illustration of the nanoparticles; (B) principle of intensified SDT; (C) in vivo self-oxygen production principle schematics. Reproduced (adapted) with permission.126 Copyright 2017, American Chemical Society.

In many cases, sonochemical reaction further potentiates tumor resistance, leading to malignant cell proliferation and drug resistance126 against SDT. These negative issues can be tackled by combining hypoxia-causing drugs with nanoparticles. For example, TPZ (a hypoxic cytotoxin drug) was loaded into mesoporous TiO2 nanoparticles, which was modified with S-nitrosothiol (R-SNO).127 Upon irradiation, TPZ was activated, followed by the generation of ROS, which is not only specific against hypoxia but also induces NO release on-demand from the SNO group for better SDT effect. To mitigate the hypoxia problems, Wang et al. developed multifunctional therapeutic nanoplatforms for delivering hematoporphyrin monomethyl ether (HMME) and acriflavine (ACF).128 The study mentioned above employed HMME as a sonosensitizer and ACF as an inhibitor of HIF-1α/HIF-1β for precise tumor targeting and tackling the adverse effects of SDT. The presented attractive nanotechnology approaches for the effective enhancement of sonodynamic therapy are summarized in Table 4.

Table 4 List of different nanomaterials in SDT-based hypoxic tumor therapy
S. No. Nanoformulation Sensitizer Cancer type Strategy Ref.
1 Lipid-stabilized microbubbles (1000 to 2000 nm) Rose bengal Pancreatic Cancer Microbubbles deliver oxygen at hypoxic sites by ultrasound waves. 178
2 Gemcitabine loaded microbubbles Rose bengal Pancreatic cancer Ultrasound triggers microbubbles to release oxygen and Gemcitabine causes hypoxia-responsive anti-cancer effect. 125
3 FC-chain-functionalized mesoporous organosilica nanoparticles (180 nm) IR780 Pancreatic cancer Ultrasound waves break the hypoxias barrier by FC chain-mediated release of oxygen to modulate hypoxia. 126
4 Tirapazamine-loaded mesoporous titanium dioxide nanoparticles with S-nitrosothiol (100 nm) TiO2 Breast cancer Ultrasound triggers the release of ROS and TPZ in the hypoxia region. Under hypoxia, TPZ induces hypoxia-specific killing. The SDT-induced ROS further could sensitize the -SNO group for the on-demand release of NO. It further sensitizes the SDT effect 179
5 Protoporphyrin and cyclic arginine–glycine–aspartic pentapeptide-loaded mesoporous organosilica-MnO2 nanoparticles (100 to 133 nm) Protoporphyrin Brain cancer MnO2 converts H2O2 into O2 and enhances SDT in hypoxia. 142


SDT is a new addition for modulating hypoxia. The potentialities in high tumor penetration depth and cost-effective strategy make SDT a successful candidate for hypoxia treatment. Till now, there exist no clear and complete ROS generating mechanisms of SDT. In general, the successful eradication of tumors requires a combinational therapy with SDT. In addition, the side effects of sonosensitizer-induced toxicity and ultrasound-induced genetic alteration are also monitored for future applications.

3.5. Nanomaterial-mediated immunotherapy of hypoxic tumors

Immunotherapy (IMT) is a recent addition to cancer treatment via the activation of the immune system (T cells) though the target receptors to fight against cancer cells. Hypoxia causes the failure of IMT mainly due to the following effects: (1) changes in immune plasticity; (2) differentiation and expansion of immune-suppressive stromal cells; (3) remodeling of the metabolic landscape to support immune privilege. For addressing hypoxia resistance, different biochemical strategies such as gene transfer,129 restoration of T-Cells,130 and vascular normalization131 have been implemented. In contrast, minimal literature is available on nanomaterial-based hypoxic IMT. Im et al. performed antigen presentation by hypoxia-responsive mesoporous silica nanocarriers with dendritic cells (DCs), which can phagocytize tumor-associated antigens (TAA) even under hypoxic conditions at the tumor site.132 IMT-based hypoxia modulation and its therapeutic results are represented in Fig. 9.
image file: d0nr06271e-f9.tif
Fig. 9 Nanomaterials for the immunotherapy of hypoxic tumors. Mode of action and in vivo evaluations: (A) schematic illustration of the mode of action: (1) after systemic administration; (2) entry of nanoparticles into the tumor region; (3) DC activation (4) TAA release from the tumor cells; (5) TAA internalization and activation of DC for maturation (6) TAA presentation; (B) in vivo tumor growth curve; (C) survival curve mice. Reproduced (adapted) with permission.132 Copyright 2019, American Chemical Society.

The recent trends in this field focus on investigating and unveiling new types of immunogenic cell death, irrespective of the destruction induced by them via hypoxia in the cancer cells. Although the works of literature give novel insights for preventing the immunosuppression effects by hypoxia, the efficiency in practical applications remains unknown. Hence, it is essential to discover novel nanomaterial-based immunomodulators and further combine with different therapeutic modalities for overcoming hypoxia.

4. Nanotechnological strategies to overcome hypoxia in tumor therapy

4.1. Nanoparticles as oxygen cargoes/oxygen modulators

As stated above, there exists an urgent need for modulating or reoxygenating TME for better therapeutic efficiencies. To date, different nanotechnological efforts have been made to overcome hypoxic resistance, including the use of classic oxygen carriers with nano-formulations and the development of novel nanomaterial-based artificial oxygen modulators. Hemoglobin (Hb-an iron-rich protein) is a well-known oxygen carrier and it is widely utilized for reliving hypoxia. In particular, it binds reversibly with oxygen at different pressures. However, the NO scavenging ability and vasoconstriction effects of Hb limit its usage and application areas.133 Recently, several nano-sized Hb-loaded liposomes with fewer side effects and enhanced circulation half-life have been developed. For instance, Hb encapsulated nano-sized liposomes (200 nm) showed enhanced PDT efficacy in colon cancer,134 as depicted in Fig. 10. Hypoxia is tackled through the stable supply of oxygen from Hb, indocyanine green (photosensitizer) transfers energy from the lasers to oxygen, and then, cytotoxic ROS is formed to fight against the cancer cells. A similar manner of Hb-based oxygen delivery to the hypoxic region is performed in chemotherapy. Furthermore, the O2 interference capacity increased the drug uptake of hypoxic cancer cells, inducing a remarkably increased toxicity of the drug against the cancer cells. Interestingly, it showed significant nanoparticle internalization and accumulation in the cancer cells compared to DOX-loaded liposomes without Hb (DL).135
image file: d0nr06271e-f10.tif
Fig. 10 Nanoparticles in the delivery of oxygen carrier and oxygen modulator. Schematic illustration of the working principle and ROS generation of Hb-loaded liposomes. (A) The alleviation of tumor hypoxia and enhanced PDT; (B) ROS generation of the liposome in normoxia conditions; (C) ROS generation of the liposome in hypoxia conditions. Reproduced (adapted) with permission.134 Copyright 2018, Elsevier.

Despite its biocompatibility and efficient oxygen carrier, the short half-life and vasoconstriction effects of Hb still cause significant lag in clinical applications. Therefore, cytocompatible cargoes with superior properties are recommended to be designed and developed. As an alternative to Hb, perfluorocarbons (PFCs) and their derivatives can be used as active oxygen carriers in hypoxic tumor therapy due to their quick hydrolysis and high solubility.136 Moreover, the higher oxygen-carrying ability of the PFCs makes it more beneficial for tackling hypoxia resistance. On this basis, Cheng et al. firstly demonstrated that PFCs have excellent ability for the enhancement of the ROS levels and the inhibition of tumor growth in colon and breast cancer cells in the PDT process.137 Recently, PFCs were encapsulated into albumin in the colon and breast cancer mice model. After the successful injection of nanoparticles, the first and second stages of oxygen delivery were in progress along with the PFCs and red blood cell infiltration. Song et al. decorated TaOx nanoparticles with PEGylated PFC nanodroplets for treating hypoxic breast cancer through RT.138 PFC acted as a reservoir in order to modulate the oxygen concentration.

Though hypoxia modulation can be implemented using hyperbaric oxygen therapy or via oxygen carriers in many ways, their output and reproducibility remain low and limited.139,140 Moreover, artificial oxygen carriers are valid only at a short-range and are not effective in distanced tumor cells, which are located far from the intratumor blood vessels. For these reasons, in situ oxygen production techniques are developed. Due to the biocompatible and non-toxic characteristics, the ability of catalysis, and high reactivity and specificity, the catalytic activity-incorporated MnO2 nanoparticles were implemented for performing the conversion of overexpressed H2O2 molecules into oxygen in a hypoxic environment.124 For example, MnO2 nanoparticles were entrapped into the polyelectrolyte–albumin complex to modulate the TME.141 The results showed an increase in oxygen level up to 45% in the tumor and the corresponding tumor pH from 6.7 to 7.2. In addition, improved downregulation of HIF-1 and VEGF in the tumor cells was observed. Similarly, multifunctional MnO2 nanoparticles were developed by integrating mesoporous organosilica with protoporphyrin and cyclic arginine–glycine–aspartic pentapeptide for treating brain cancer.142 In this system, MnO2 behaved as an inorganic nanoenzyme for decomposing endogenous H2O2 molecules into oxygen and for further overcoming the GSH-mediated consumption of oxygen in the following reactions.143

 
MnO2 + 2H+ → Mn2+ + H2O + 1/2O2(1)
 
MnO2 + H2O2 + 2H+ → Mn2+ + 2H2O + O2(2)
 
MnO2 + GSH → Mn2+ + GSSG(3)

Recently, hydrophilic terpolymer-albumin-based MnO2 nanoparticles144 and polyelectrolyte–albumin based MnO2 nanoparticles were also prepared for reoxygenating hypoxic breast cancer cells.145 The practical findings showed that a single dose of NPs (90 μM MnO2) can consistently generate O2 for a minimum of six cycles during every 30 min of radiotherapy. The rapid diffusion of H2O2 and protons across the polyelectrolyte–albumin complex and reactive sites of the MnO2 cores were credited with this O2 generation process. Moreover, the consumption of Hb ions and the formation of Mn-oxo-hydroxide led to a change in the tumor pH,146 depicting a sustained increase in the pH value under hypoxic conditions. In most cases, MnO2 nanoparticles were used with other agents such as radiosensitizers, photosensitizers, or sonosensitizers for cancer therapy. For instance, MnO2 nanoparticles were integrated with high-Z element gold nanoparticles to tackle tumor hypoxia in radiation therapy.79 The gold core acted as the sensitizer and the MnO2 shell triggered the conversion of H2O2 into O2 to overcome hypoxia-associated RT resistance. In addition, the MnO2 NPs could release Mn2+, which was realized in imaging-guided therapy. Similarly, a catalase-loaded tantalum oxide bioreactor was developed for the conversion of H2O2 into H2O and O2.147 The results showed an efficient conversion of H2O2 in the TME and an improvement in the oxygenation of the tumor in the hypoxic region. As a result, a remarkable synergistic RT sensitization effect was achieved with the in vivo treatment of animals at a dose rate of 8 Gy min−1.

Irrespective of their efficiency in overcoming hypoxia, MnO2 and other nanoparticles are simply limited by the finite H2O2 availability because the cells are not an infinite reservoir of H2O2. In addition, they lag in cytocompatibility issues due to the employment of several toxic chemicals during the synthesis, thereby recommending the development of nanoparticles via a biosynthetic method. However, the biosynthetic process has its own limitation in MnO2 preparation,148 where the scalability and reproducibility are still under question.

4.2. Nanoparticles as hypoxia-activated prodrug-delivery cargos

Using the hypoxic microenvironment, hypoxia targeted tumor modalities such as prodrug actuation,149,150 hypoxia selective gene therapy,151 HIF-1α inhibition therapy,119 hypoxia-responsive groups,152 and inhibition of signaling pathways153 are employed. These hypoxic prodrugs are non-toxic at typical oxygen concentration, whereas they undergo structural transformation under hypoxic conditions and then capture hydrogen atoms from the surrounding biomacromolecules, resulting in cell death. In most situations, hypoxia-activated prodrugs are used in PDT to improve the therapeutic efficiencies. However, the adequate enrichment of photosensitizers and hypoxic prodrugs in the tumor environment are essential issues that need to be rectified.

Guo et al. developed novel nanoparticles, which were embedded with an angiogenic vessel-targeting peptide for hypoxia-activated prodrug release with the assistance of phototherapy.154 As represented in Fig. 11, the ROS-mediated anti-cancer effect was initially achieved through PDT and TPZ was activated consecutively by intense hypoxia to produce cytotoxic radicals. More importantly, TPC (5-(4-carboxyphenyl)-10,15,20-triphenyl chlorine) was used as a photosensitizer so as to induce photochemical oxygen depletion in the PDT process, thus creating a hypoxic microenvironment and promoting the upregulation of VEGF and angiogenesis. The hypoxia-specific peptide accelerated the accumulation of the nanoparticles at the tumor site and supported the PDT process. The subsequent prodrug-photo combinational treatment exhibited higher anti-tumor efficacy in cellular and animal studies. It implies that the hypoxia-mediated promotion of angiogenesis could benefit as an innovative modality for hypoxic tumor therapy. TPZ was also loaded into membrane camouflage nanoparticles with indocyanine green photosensitizer against breast cancer,155 in which laser illumination produced ROS, which further resulted in severe oxygen consumption. At this stage, hypoxia got intense and the structure of the hypoxic prodrug was altered for synergistic therapy. The results of the in vivo studies showed 1.3 times higher (64%) tumor inhibition rate than that using PDT alone. In addition, the membrane camouflage system enables less macrophage clearance so as to obtain a higher cell death and hypoxic environment. In a similar way, TPZ with a photosensitizer (IR780) were loaded into HA nanoparticles towards bladder cancer.156 The study demonstrated that the simultaneous oxygen consumption mechanisms of photothermal and photodynamic reaction-activated TPZ could be connected with vascular shutdown and singlet oxygen generation. More recently, the platinum (from IV to II) and TPZ-formed polyprodrug nanogels extended the NADPH oxidases’ appearance to induce oxygen consumption and boost ROS formation.157 The exaggerated hypoxia environment additionally activated TPZ to yield cytotoxic radicals. Moreover, the intelligent nanostructure improved the release of the redox-sensitive drug and prolonged the circulation in a mouse model. Even though TPZ is an efficient hypoxia-selective bio-reductive agent in in vitro studies, clinical trials did not demonstrate the potentiality and showed modest toxicity.


image file: d0nr06271e-f11.tif
Fig. 11 Nanoparticles in hypoxia-activated prodrug release. (A) Schematic illustration of PDT-induced hypoxia and the promoted angiogenesis. Reproduced (adapted) with permission.154 Copyright 2017, Elsevier.

Apart from TPZ, banoxantrone, NLCQ-1, and dinitrobenzamide mustards can also be utilized as HAP agents to overcome hypoxia resistance. Zhang et al. reported a co-delivery system of banoxantrone and DOX to improve photochemotherapy efficiencies.158 Metal–organic particles from copper (Cu(II)) facilitated a deeper penetration in the solid tumors via microvesicle-mediated intercellular transfer, followed by PDT to improve the chemotherapy. Banoxantrone is quite efficient compared to other HAPs because it simply exhibits toxicity in the presence of oxygen. Hence, the feasibility of new routes should be examined and well-evidenced by biocompatible surface modifications and size tuning along with banoxantrone loading. The mentioned agents have been established in early clinical development stages with limited nano-based formulations. These findings suggest that mixed administrations of hypoxia-responsive prodrugs and hypoxia-activated anti-tumor agents could serve as a promising strategy in order to realize efficient hypoxia tumor therapy. Despite the significant developments in hypoxia-associated tumor therapy, limitations still exist due to the moderate vasculature and oxygen supply at the tumor site. However, compared with oxygen-dependent approaches, the hypoxia-activated prodrug activation strategy suggests the novel and effective eradication of hypoxic tumors. Compared to oxygen modulation techniques, the prodrug activation strategy has more advantageous as it works effectively only under low oxygen concentrations.

4.3. Nanoparticles as HIF-1 inhibitor-delivery cargos

HIF-1 is the main transcription factor triggered under the hypoxic environment. It can activate an array of target genes towards the modulation of various cellular responses, including proliferation, apoptosis, angiogenesis, and metabolism. The upregulated HIF-1 level often contributes to the failure of cancer treatment by vascular reconstruction.159 Accordingly, researchers found that the inhibition of signaling pathway activity and activation mechanisms of HIF-1 could suppress the tumor growth, making it a suitable strategy for hypoxic tumor treatment. However, these small molecular inhibitors are immediately eliminated from the blood after intravenous injection, leading to low accumulation in the tumor and poor inhibition efficiency. HIF-1 loaded nanoparticles with better therapeutic abilities are needed. For instance, it is proved that the hindrance of angiogenesis and mitochondrial apoptosis by using dual inhibitors of celastrol and axitinib-loaded mesoporous silica nanoparticles (120 nm).160 The inhibition results were obtained from the raised grades of caspase-3 and PARP; meanwhile, the appearance of CD31 and Ki-67 in IHC analysis was diminished. These results implied the great potential and scope of integrating the dual delivery modality for treating cancer, specifically in uncontrollable cases.

Although many small-molecule inhibitors of HIF-1 have been identified, their targeting ability remains unsolved and small interfering RNA (siRNA) silencing remains a hot topic. Bartholomeusz et al. delivered siRNA into the tumor cells with pristine single-walled carbon nanotubes (SWCNTs).161 The experimental results showed enhanced inhibition of the HIF-1 activity and a low protein scale of nearly 70% to 80% since a majority of siRNA were dissociated from the SWCNTs inside the cells, in which few siRNA maintained their RNAi activity and was still intricated with the nanotubes. This problem needs to be addressed. Recently, Liu et al. developed a polymer-based cationic micelle nanostructure (58 nm) for siRNA delivery in prostate cancer cells.162 The results showed enhanced cell proliferation inhibition, restrained cell migration, and disturbed angiogenesis under hypoxic mimicking conditions. In addition, the results of in vivo experiments showed a downregulation of the MDR1 gene expression and inhibition of tumor growth without the activation of the innate immune responses.

As depicted in Fig. 12, Zhao et al. demonstrated a co-delivery system of siRNA and the anti-cancer drug via the polymeric nanostructure for the hypoxic pancreatic cancer.163 The results revealed an enhanced stability and prolonged blood circulation with enhanced synergistic in vivo anti-tumor effects. More importantly, siRNA showed an enhanced innate immune suppression, thus suggesting its use in a broad array of tumor treatments by the co-delivery of siRNA and chemotherapy drugs. Similarly, with the help of lipid-calcium-phosphate nanoparticles, HIF1α-siRNA-combined PDT is performed to overcome hypoxia resistance in neck and head cancer treatments.164 The results suggested that the downregulation of HIF1α can boost ROS formation in the cancer cells towards better apoptotic action. The combined silencing HIF1α gene therapy could effectively enhance the PDT efficiencies. More recently, the HIF-1α inhibitor (acriflavine)-functionalized nanometric radiosensitizer (yolk–shell Cu2−xSe@PtSe) was applied in radiation therapy for breast cancer cells.165 The nanoparticles not only evidenced the ability to relieve hypoxia by arresting the G2/M phases of the tumor cells but also exhibited a strong attenuation of the X-rays due to their composition of high Z elements.


image file: d0nr06271e-f12.tif
Fig. 12 Nanoparticles in the HIF-1 inhibitor cargo. Schematic representation of the synthesis and action principles of the HIF-1 inhibitor: (A) nanoparticle preparation; (B) in vivo nanoparticle uptake. I: siRNA protection from degradation. II: Nanoparticle ensures stability and circulation in the bloodstream. III: The co-delivery of si-HIF1a and Geminto cancer cells. Reproduced (adapted) with permission.163 Copyright 2014, Elsevier.

To date, many strategies centering on HIF-1 for medicine production have been reported.166,167 However, the compatibility, chemical integrity, variable clinical effects, and clinical usage of such strategies are still undergoing fluctuations, thus limiting their usage. In recent years, silver nanoparticles (AgNPs) have also been reported to alter the mitochondrial membrane potential of the cancer cells by the induction of ROS. Yang et al. demonstrated the ability of AgNPs to inhibit tumor maturation by suppressing the HIF-1α activity.168 Great insight into inhibition mechanism was obtained by the results even under hypoxic conditions. AgNPs also showed the inhibition function of angiogenesis and the downregulation of VEGF-A and GLUT1. Although satisfactory inhibition activity was demonstrated with silver nanoparticles, the working mechanism is still not precise. Specifically, studies of enzymatic degradation and absorption–distribution–metabolism–excretion (ADME) mechanism have to be conducted for future applications as it is metal-based and can be easily degraded by the body enzymes. Besides, compared with oxygen modulation and prodrug activation strategies, the inhibitor-based strategy has better therapeutic efficiency as it is oxygen-independent.

5. Challenges and perspectives

In conclusion, we have reviewed the most recent and relevant research activities, which offers great help to the researchers in tackling hypoxia-associated therapeutic resistance. Unfortunately, most of the developments and strategies are still at their infancy for overcoming hypoxia. We have highlighted the overall strengths and weaknesses in the recent nanotechnology-based strategies for effective hypoxia tumor eradication. This article provides complete solutions for researchers who are seeking to overcome the hypoxia barrier in cancer therapy. It is clearly understood in our literature survey that there still exists a great need for the adequate exploration of TME in the context of hypoxic tumor therapy regardless of their oxygen concentration and pH levels.

On the other hand, all the listed approaches including RT, Chemo, PDT, SDT, and IMT have demonstrated their remarkable capabilities for improving oxygenation in the tumor tissues, which becomes an essential component to clarify the spatial association between the distribution of oxygen molecules and hypoxic tumor regions as well as to optimize the time for post-reoxygenation in radiotherapy. It is clearly understood that radiosensitizer-loaded nanoparticles promise better evaluations for cancer treatments due to their stable half-life and effectiveness. In addition, nanomaterials with high-atomic number elements are also employed to increase the radio-sensitivity and to attenuate the hypoxia. For the sake of this issue, recent innovations have been focused on the development of new multifunctional and specific-structured nanomaterials. Nevertheless, it is vital to increase the cellular uptake, reduce non-specific recognition, and accelerate renal clearance of the sensitizer to avoid long-term body retention, which may induce further side effects. It is therefore essential to develop nano-radiosensitizers with enhanced renal clearance and biocompatibility in a broad scope in the future. Chemotherapy is quite different in hypoxia tumors as it involves a combination of other therapeutic modalities.

Furthermore, chemotherapy uses oxygen modulation techniques and hypoxia-activated drug delivery principles. Compared with other conventional polymeric nanoparticles, MnO2-based nanoformulations are widely employed as drug carriers due to their excellent biocompatibility. MnO2-based nanoformulations are also used as cargos of other therapeutic sensitizers. Chemoradiation therapy is one of the most frequently combined modalities that require the simultaneous activation of hypoxia-active prodrugs and radiosensitizers.

With respect to chemoradiation, one of the biggest challenges in treating hypoxic tumors is the complete cellular uptake and excretion of nanoparticles. Despite this, there is no complete explanation of the ADME mechanism in chemotherapy. Thus, the exploration of ADME mechanism in hypoxic cells is necessary in future research. In addition, it is noted that novel nanoparticles should be developed, which have fine-tuned tiny size, can penetrate the hypoxia region for better targeting, and have bioavailability to achieve better efficiency in chemotherapy. The lower oxygen concentration, the limited penetration depth of light, undesired toxicity of PS towards healthy tissues upon light irradiation, as well as the site-specific delivery of PS to deep tumor tissues or sub-cellular organelles for enhanced toxicity are significant challenges in PDT. Therefore, intelligent oxygen modulation systems and multifunctional oxygen carriers, which can synergistically overcome these systemic barriers, are required for maximum anti-cancer efficacy. More importantly, the PDT-induced side consequences such as the invasion and metastasis of cancer cells should be seriously considered along with the biocompatibility, biodegradability, tractability, and reproducibility of oxygen modulators. SDT is a non-invasive and cost-effective modality in cancer treatment and also performs reoxygenation, H2O2 conversion, and use of hypoxia-responsive drugs to tackle therapeutic resistance. In most of the cases, SDT is performed with microbubbles with the aid of external ultrasound. The sustainable release and consistency of microbubbles in oxygen modulations are still at their infancy, which further need several improvements for attaining complete tumor eradication. Herein, it is crucial to adopt novel two-dimensional nanostructures in combination with sonosensitizers as well as oxygen modulators. The recent addition to cancer therapeutics is IMT. However, it is still not effectively performed against hypoxic environments due to the abnormal vascularization and cellular functionalities, which is indifferent to immune response. It significantly provides the basis of a new cancer treatment option, whereas the therapeutic options that exist are extremely limited. Hence, it is recommended to combine IMT with other modalities and novel immunomodulators, which could work even under the hypoxic environment, need to be developed.

In nanomaterial-based strategies, oxygen modulation techniques are widely performed. In particular, Hb and PFCs show great potential in hypoxia tumor therapy as oxygen carriers. Their release is through a simple diffusion mechanism that limits the effectiveness and a sustained release requires external stimuli. Different kinds of strategies, such as light- or ultrasound-triggered release, are employed to tackle these challenges. More inventions or validations regarding the synthesis of multifunctional nanostructures together with the use of pH levels in TME and receptors of hypoxic cells are needed to be carried out. It is recommended to focus on the potential nanostructures with excellent biocompatibility and superior properties, metal–organic frameworks, and two-dimensional nanostructures (e.g., black phosphorus and graphene) in the future studies since they can be easily functionalized and have a large surface area to volume ratio for loading oxygen and therapeutic agents. As an alternative to PFCs, MnO2 nanoparticles have substantially shown their potential in tumor reoxygenation. However, finite H2O2 supply in the cells leads to a reduction in the conversion performance of MnO2 nanoparticles.

In addition, the use of free Mn2+ ions as imaging agents for MRI in hypoxia gives a new path for the investigation of new-generation efficient hypoxia tumor theranostics. It is also advisable to develop biomimetic nanoenzymes that are capable of performing efficient catalysis with enhanced penetration to hypoxic cells. Since hypoxia modulation drugs show some hurdles in clinical application, including toxicity and other side effects, hypoxia-causing drugs are imported as substitutes. We must to pay proper attention for developing future drugs and nano-formulations that can promote better clinical trials without side effects. Besides, the hypoxia-causing activation mechanism, nanoparticle-transmission mechanism, and delivery mechanism into far cells in the hypoxia environment need to be unveiled. Among various strategies, HIF-1 inhibition has become a hot topic, specifically the Si-RNA mechanism. Furthermore, the inhibition of other receptors and downregulation of angiogenesis pathways have also been used for cancer therapy but the mechanism is not yet explained very well. Besides, studies for the better efficiency of cancer therapy are still required. There still exists plenty of room to be explored in precisely predicting RNA silencing in hypoxic cells. In a nutshell, the current review opens up new doors for the future research of nanomaterial-enabled hypoxia tumor therapy.

Author contributions

Zhongjian Xie, Guiqing Wang, Ni Xie, Joice Sophia Ponraj, and Han Zhang provided the whole concept and wrote the abstract, challenges, and perspectives. Aravindkumar Sundaram and Ling Peng wrote all the other sections. Luxiao Chai modified the grammar. Xiangjiang Wang, Bin Zhang, Guohui Nie, and Rajesh Kumar Manavalan gave suggestions for the modification. All the authors contributed to the general discussion.

Conflicts of interest

The authors declare no conflict of interest.

Acknowledgements

The author J. S. Ponraj kindly acknowledges DST-INSPIRE Faculty Scheme (DST/INSPIRE/04/2016/000292), SERB-EMR (EMR/2017/004764) and EU-EC/MSCA-COFUND-2015-FP Nano TRAIN for Growth II No.: 713640 for the financial supports. The research is partially financially supported by the Science and Technology Development Fund of Macao Special Administration Region (SAR) (No. 007/2017/A1 and 132/2017/A3), National Natural Science Fund (Grant No. 61875138, 61435010, and 6181101252), and Science and Technology Innovation Commission of Shenzhen (KQTD2015032416270385, JCYJ20150625103619275, and JCYJ20170811093453105). The authors also acknowledge the support from Instrumental Analysis Centre of Shenzhen University (Xili Campus). One of the authors (Rajesh Kumar Manavalan) thanks the contract no. 40/is2.

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Footnote

These authors equally contributed to this work.

This journal is © The Royal Society of Chemistry 2020