Injectable Hydrogels for Localized Cancer Therapy

Traditional intravenous chemotherapy is relative to many systemic side effects, including myelosuppression, liver or kidney dysfunction, and neurotoxicity. As an alternative method, the injectable hydrogel can efficiently avoid these problems by releasing drugs topically at the tumor site. With advantages of localized drug toxicity in the tumor site, proper injectable hydrogel as the drug delivery system has become a research hotspot. Based on different types and stages of cancer, a variety of hydrogel drug delivery systems were developed, including thermosensitive, pH-sensitive, photosensitive, and dual-sensitive hydrogel. In this review, the latest developments of these hydrogels and related drug delivery systems were summarized. In summary, our increasing knowledge of injectable hydrogel for localized cancer therapy ensures us that it is a more durable and effective approach than traditional chemotherapy. Smart release system reacting to different stimuli at different time according to the micro-environment changes in the tumor site is a promising tendency for further studies.


INTRODUCTION
With the deterioration of the environment, the incidence of cancer is increasing year by year. In 2018, there were 18.1 million new cancer cases worldwide (9.5 million males and 8.6 million females), and the death toll was 9.6 million (5.4 million males and 4.2 million females) (Bray et al., 2018). The global cancer burden is further aggravated. One in five men and one in six women worldwide will develop cancer, and 1 in 8 men and 1 in 11 women will die for cancer (Bray et al., 2018;Sivaram et al., 2018).
Different strategies and methods are applied for cancer patients according to their different diagnose and stage. Possible therapies include chemotherapy, radiation, surgery, immunotherapy, targeted therapy, and gene therapy Roy and Trinchieri, 2017;Bykov et al., 2018;Senft et al., 2018). Among those options, chemotherapy plays a pivotal role in tumor control and prevention of recurrence. The anti-tumor activity of chemotherapeutic drugs kill tumor cells with its drug toxicity (Zhou et al., 2017). Without selective or targeted killing, normal tissues could be harmed by chemical drugs along with tumor cells (Andreyev et al., 2014).
With the development of hydrogel, traditional chemotherapy drugs are booming a new life. Drugs are injected within the hydrogel directly into the tumor or adjacent to it (Elias et al., 2015;Ma et al., 2015;Pan et al., 2018). Drugs could be localized in a crosslinked 3D network of hydrophilic polymer chains Bu et al., 2017). In this way, drug toxicity is limited within a localized area where tumor cells lie. Meanwhile, localized hydrogel reveals the ability for continuous efficient drug delivery in the tumor site Yang et al., 2018). To enhance this character, a variety of systems have been developed with different composition, including polyphosphazene (PPZ), polyethylene glycol (PEG), and Polylactate glycolic acid PLGA (Bu et al., 2017;Ahmad et al., 2018;Cheng et al., 2018;Gajendiran et al., 2019).
Most of the hydrogel is insoluble in water but excellent in water absorption capacity (from 10% to thousands of times their dry weight) (Bin Imran et al., 2014;Murgia et al., 2018) (Figure 1). The soft, moist surface, and affinity with the tissue greatly reduce the stimulation of the human body. Most materials that make hydrogels are non-toxic Batista et al., 2019;Gajendiran et al., 2019). According to the response to external stimuli, hydrogels can be divided into ordinary hydrogels and smart hydrogels (Gu et al., 2017). Original hydrogels are not sensitive to environmental changes (Castelletto et al., 2019;Kerdsirichairat et al., 2019;Luo et al., 2019). While smart hydrogels could be affected by pH, temperature, and photoelectricity. It results in changeable gel volume and traits for smart hydrogels (Deepa et al., 2012;Milcovich et al., 2017;Chen et al., 2019). These hydrogels are currently widely used in tissue engineering, drug delivery, and other fields (Bae et al., 2013;Del Bufalo et al., 2016;Casey et al., 2017;Castelletto et al., 2019).

THERMOSENSITIVE HYDROGELS
Temperature-sensitive hydrogels are hydrogels that change in volume as the ambient temperature changes (Klouda, 2015;Chen et al., 2018). The gel always has a certain proportion of hydrophobic and hydrophilic groups. Temperature changes can affect the hydrophobic interaction of these groups. The hydrogen bonding between the macromolecular chains causes the gel structure to change, and volume changes occur . The temperature at which the volume changes is referred as lower critical solution temperature (LCST) (Sapino et al., 2019). Under this temperature, the gel swells in aqueous solution. Once the temperature rises to LCST, the gel shrinks (Lei et al., 2012;Wang et al., 2015). Its unique properties can be used as a drug carrier, which is injected into the body after being combined with a drug at a low temperature (Le et al., 2018). Forming a colloidal state with the help of body temperature makes it a drug sustained-release system, which simplifies not only medical treatment but also patients' suffering . Main thermosensitive injectable hydrogels include chitosan/glycerophosphate (C/GP), hyaluronic acid (HA), PLGA based hydrogel, PEG-based hydrogel, PECE, and PECT (Guo et al., 2012;Klouda, 2015;Huang et al., 2016;Le et al., 2018;Sapino et al., 2019). Their characters and drug delivery systems were summarized in Table 1.
In one study (Huang et al., 2016), injectable thermosensitive doxorubicin (DOX) delivery system was developed with PECT hydrogel. Instead of hydrogel based on free DOX diffusion, which suffered from rapid drug clearance and poor drug penetration in tumor tissue, self-polymerized drug-loaded nanoparticles were encapsulated into PECT hydrogel (Huang et al., 2016). After in vivo injection, PECT gel exhibited a transition phase between sol and gel. The viscosity increased abruptly once the  temperature is higher than 28 • C. With the highest viscosity at 37 • C, the hydrogel turned to gel from sol (Figure 2). Loaded nanoparticles were dissociated from hydrogel and diffused within tumor tissue by EPR effect. Intracellular chemical drug release limited its toxic effects and enhanced its anti-tumor effectiveness. Contrasted with intravenous drug injections (I.V.), a thermosensitive hydrogel with nanomedicine loaded is an efficient drug delivery system, which enabled continuous drug release around tumor tissues (Huang et al., 2016). Two or more elements loaded in one thermosensitive hydrogel has emerged as a promising drug delivery system for its superior anti-tumor efficacy. Polo-like kinase 1 (PLK1) gene is recognized as a key regulator of tumor cell meiosis and mitosis (Ma et al., 2014). RNA interference-based on PLK1shRNA can specifically reduce the function of the target gene in the tumor. A strategy of DOX and PLK1shRNA/PEI-Lys co-delivery hydrogel was developed for the treatment of osteosarcoma (Ma et al., 2014). In this method, PLK1shRNA/PEI-Lys in the hydrogel can greatly enhance the anti-tumor effect of DOX. With the synergistic effect from PLK1shRNA/PEI-Lys and DOX, significant osteosarcoma apoptosis was caused by the co-loaded hydrogel (Figure 3) (Ma et al., 2014).

PH-SENSITIVE HYDROGELS
Glycolysis of tumor cells can cause acidification in the environment next to tumor tissues, resulting in lower pH value in the extracellular matrix than normal tissues (Kenney et al., 2018;Hu et al., 2019). A pH-sensitive hydrogel is a polymer gel in which the volume of the hydrogel changes depending on the pH of the external environment and the ionic strength (Liao et al., 2017;Liu et al., 2019). Such gels contain a large number of readily hydrolyzable or protonated acids, base groups such as carboxyl groups and amino groups (Lym et al., 2016). The dissociation of these groups is affected by the external pH. When the external pH changes, the degree of dissociation of these groups changes correspondingly, causing the internal and external ion concentration to change (Norouzi et al., 2016). In addition, the dissociation of these groups will destroy the corresponding hydrogen in the gel. The bond reduces the crosslinking point of the gel network, causing a change in the structure of the gel network and the degree of swelling of the hydrogel (Qu et al., 2017;Oroojalian et al., 2018). With this property, the rate of diffusion and release of the drug in the gel can be conveniently adjusted and controlled (Samanta et al., 2015). A variety of elements for pH-sensitive hydrogel were explored in the past decades. Their characters and drug delivery systems were summarized in Table 2. One of the choices is based on chitosan-grafted-dihydrocaffeic acid (CS-DA) and oxidized pullulan (OP) (Liang et al., 2019). With classical drug for antitumor therapy, DOX-loaded hydrogel was tested to explore its reactions for the pH changes in the tumor environment. With glycolysis in the tumor site, a decrease of pH triggered the drug release (Liang et al., 2019). Compared with the morphology of hydrogel at pH 7.4, significant disintegration of hydrogel resulted in larger pore size at pH 5.5 (Figure 4) (Liang et al., 2019). After 60 h at pH 5.5, more than 80% of DOX was released from the hydrogel. The hydrogel was co-cultured with Hct116 cells (colon tumor cells) to test its anti-tumor effect (Liang et al., 2019). DOX is continuously stable released from hydrogel at pH 5.5 and 7.4. In both conditions, DOX can be effectively released for more than 3 days (Liang et al., 2019).
In recent years, aspirin has been found to inhibit carbon monoxide synthase, inhibit nitrite-mediated DNA damage, reduce surviving, inhibit nuclear transcription factors, proteasomes, and calcium-activated neutral proteasome genes by inhibiting cyclooxygenase (Lu et al., 2008;Choi et al., 2013). The expression and other mechanisms play an anti-tumor effect. After loading aspirin into the hemicellulose hydrogel, it was found that 85% of the drug could be released continuously for 5-6 h under pH 7.4 (Choi et al., 2013). Sun et al. successfully prepared a series of acrylic acid and acrylamide copolymer grafted hemicellulose hydrogels by free radical polymerization (Sun et al., 2013). The combination of aspirin and the drug showed that the release rate of the drug in the simulated gastric juice was slower, and the release rate in the simulated intestinal fluid was significantly faster than that of the simulated gastric juice. When the release time was 12 h, the cumulative release rate reached 90%, which shows excellent sustained release properties (Sun et al., 2013).
In addition, Wang et al. first inserted dexamethasone phosphate into molecularly imprinted polymer nanospheres and loaded the polymer onto a pH-sensitive hydrogel, making it a biosensor that inhibits inflammation (Wang et al., 2010). Since the inflammatory reaction can lead to an acidic environment, this pH-sensitive hydrogel can rapidly release the drug at pH 6.0∼7.4 to inhibit the inflammation (Wang et al., 2010). This different method is resistant to pH-sensitive hydrogels. The application of oncology drugs has brought new ideas.

PHOTOSENSITIVE HYDROGELS
The mechanism of a light-sensitive hydrogel is divided into two types according to the properties of the photosensitive material (Chang et al., 2019). One is to directly add the photosensitive molecular material to the temperature-sensitive gel, and convert the light energy into heat energy to make the temperature inside the gel reach the phase transition temperature. In this way, hydrogel produces photosensitivity. Another kind of chromophore is introduced into the gel structure (Norouzi et al., 2016). The physicochemical properties of the chromophore are changed when subjected to light stimulation. By changing the network structure, hydrogel macroscopically exhibits photosensitivity. Usually, a structure such as azobenzene, spiropyran, o-naphthoquinone, anthracene, nitrophenyl, and coumarin is introduced into the gel (Tam et al., 2017). Among them, ruthenium, nitrophenyl, and coumarin compounds mainly take advantage of photo-cleavage photosensitive groups, which are bonded to the hydrophobic end through an aryl methyl bond (Norouzi et al., 2016;Tam et al., 2017). Under ultraviolet light or near-infrared light, the ester group is broken. The photosensitive reaction is caused. The hydrophobic end is converted to a hydrophilic end, causing the gel to dissociate. The azobenzene compound is controlled by the conversion of the cistrans structure. Their characters and drug delivery systems were summarized in Table 3.
One of the applications for photosensitive hydrogels is the platform for cell culture and 3D tumor micro-environment studies. With advantages of its photosensitive character, cell detachment on the surface of hydrogel was done layer-by-layer to form a 3D cell culture medium (Figure 5) (Wang et al., 2014). Photoinitiated copolymerization of P (OEGMA-co-VDTco-SPAA) (POVSP) hydrogels happened with UV irradiation. The compressive strengths of hydrogels were up to 5.1 MPa, which is strong enough for cell culture (Wang et al., 2014). It is revealed that photosensitive hydrogel is suitable for 3D cell culture model, which is vital for the study of the mechanism for tumor development.
To achieve the same purpose, a photocleavable terpolymer hydrogel was developed as the basic technique for 3D bioprinting. This hydrogel is capable of self-shaping directly to the UV irradiation. It is designable by using selective illumination to UV light with the specific area covered with darkness (Figure 6) (Liao et al., 2015). The printable hydrogel is an inspiring design for controlled drug delivery with district distribution. It is a key technique for the realization of 4D drug delivery with both dimensions of time and space. With drugs loaded in the 3D space of hydrogels, dynamic drug release can be realized. In this process, different drug could be controlled to be released in different time with purpose (Xu et al., 2015;Kim et al., 2017;Guo et al., 2018). This is the typical way of 4D drug delivery with additional dimensions of time.

DUAL-SENSITIVE HYDROGELS
With the increasing requirements for the precision of controlled release of drugs, multi-sensitive hydrogels have received more and more attention (Bardajee et al., 2017). In particular, cosensitive hydrogels for temperature and pH is widely researched. Temperature and pH are two important factors in physiological, biological, and chemical systems (Bardajee et al., 2017;Fathi et al., 2019). The temperature-pH double-sensitive hydrogel consists of a temperature-sensitive and pH-sensitive two-part hydrophilic polymer network (Lym et al., 2016). Usually formed with two or more monomers or polymers, which respond to temperature and pH, respectively.
The combination of temperature and pH sensitivity is crucial for the management of locoregional tumor recurrence (Mackiewicz et al., 2019). A novel pH-sensitive thermosensitive hydrogel loaded with modified doxorubicin-based prodrug nanoparticles (PDNPs), which is more efficient for tumor management than free DOX . Good biocompatibility and anti-tumor activity were verified by  Lung Cancer DOX and amoxicillin co-loaded hydrogel response to pH decrease. The hydrogel is an ideal system for mucosa-localized tumor and infection management Guo et al., 2018 Frontiers in Chemistry | www.frontiersin.org  in vitro uptake and cell toxicity. For in vivo experiment, 4T1 cells with luciferase-tagged expression were implanted into mice. Management by temperature and pH co-sensitive hydrogel was remarkable (Figure 7) . It is a promising strategy for preventing the locoregional recurrence of the tumor. Co-sensitive hydrogel with dual photoluminescence was developed with PL and PNIPAM . This hydrogel contains a core which was made up of a red-emission complex and a blue-emission d-TPE. This nanoparticle is sensitive to the change of temperature and pH . This hydrogel is stimulated by both temperature and pH and is more adaptable to the complex environment of human body fluids. In addition, the application of two or more materials, through their interaction, not only can improve the mechanical strength of the hydrogel, but also improve the precision of controlled release. With this character, the stimuli-responsive hydrogel has a wide application in medical imaging, cancer diagnosis, and advanced antitumor drug delivery .

CONCLUSIONS
The unique character of hydrogel makes it an efficient functional medium for drug delivery Fathi et al., 2019;Mackiewicz et al., 2019). Given the limits from chemical drug toxicity for normal tissue and organs, localized drug delivery system by hydrogel has been a crucial method for cancer management. Related studies mainly focused on the delivery function and the methods of stimuli-response (Lym et al., 2016;Bardajee et al., 2017;Wei et al., 2017;Fathi et al., 2019). The smart hydrogel was developed with accurate responses to tiny changes in temperature, pH, and light. For now, drugs can be easily delivered to cancer tissue at the right time point. In the future, co-loaded drugs, including DNA, RNA, protein, and related products, would be a key point. The constantly accurate drug delivery system can realize anti-tumor drugs release followed by tissue repair factors. In this way, demission of time and space for drug delivery would be mixed in one hydrogel, making it a 4D functional hydrogel. It can make hydrogel a perfect choice for local chemotherapy and cancer management.

AUTHOR CONTRIBUTIONS
All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.

ACKNOWLEDGMENTS
The financial support from Ministry of Science and Technology of China (2016YFB1101501) is gratefully acknowledged.