Recent Advances and Impact of Chemotherapeutic and Antiangiogenic Nanoformulations for Combination Cancer Therapy

Traditional chemotherapy, along with antiangiogenesis drugs (combination cancer therapy), has shown reduced tumor recurrence and improved antitumor effects, as tumor growth and metastasis are often dependent on tumor vascularization. However, the effect of combination chemotherapy, including synergism and additive and even antagonism effects, depends on drug combinations in an optimized ratio. Hence, nanoformulations are ideal, demonstrating a great potential for the combination therapy of chemo-antiangiogenesis for cancer. The rationale for designing various nanocarriers for combination therapy is derived from organic (polymer, lipid), inorganic, or hybrid materials. In particular, hybrid nanocarriers that consist of more than one material construct provide flexibility for different modes of entrapment within the same carrier—e.g., physical adsorption, encapsulation, and chemical conjugation strategies. These multifunctional nanocarriers can thus be used to co-deliver chemo- and antiangiogenesis drugs with tunable drug release at target sites. Hence, this review attempts to survey the most recent advances in nanoformulations and their impact on cancer treatment in a combined regimen—i.e., conventional cytotoxic and antiangiogenesis agents. The mechanisms and site-specific co-delivery strategies are also discussed herein, along with future prospects.


Introduction
Cancer is one of the leading causes of death, and its treatment remains one of the most severe challenges worldwide. Globally, about 1 in 6 deaths is due to cancer. The World Health Organization (WHO) projected that cancer was responsible for an estimated 9.6 million deaths in the year 2018. The number of global cancer deaths is currently projected to increase by 45% between 2008 and 2030 [1]. Carcinogenesis is a complex and dynamic process, comprised of cancer-associated fibroblasts and myofibroblasts, neuroendocrine cells, adipose cells, immune and inflammatory cells, blood and lymphatic vascular networks, and an extracellular matrix (ECM). These processes altogether establish a complex cross-talk within the tumor microenvironment. Hence, cancer is described as a group of diseases characterized by uncontrolled growth/proliferation and the spread of abnormal cells [2][3][4][5][6]. Conventional treatment approaches for cancer include surgery; radiotherapy; and systemic treatments such as chemotherapy, endocrine therapy, and antiangiogenic therapy [7][8][9][10]. This paper will present the recent nanoformulation developments of anticancer (chemotherapy) and antiangiogenesis agents.
Numerous other nanoformulations of combinations with two different mechanisms of action of the drugs (e.g., anticancer/antiangiogenic drugs) in optimized ratios have also been explored. However, such systems have not been extensively clinically explored, despite their exploration in preclinical studies. The importance of combining conventional chemotherapy with antiangiogenesis can be explained through tumor vascularization, as both the tumor growth and metastasis are dependent on it. Hence, the combination therapy with these two agents can prevent the tumor recurrence and improve the antitumor effect. Nanoformulations of anticancer/antiangiogenic drugs would allow the optimization of drugs to reduce the dose-related side effects and maximize the synergy of the drugs. Recently developed nanoformulations of anticancer and antiangiogenic drugs in a nanocarrier using different approaches covered in this review are presented in Figure 1 and Table  1. Similarly, different approaches for the implementation of two drugs in a nanocarrier are depicted in Figure 2. Hence, this review explores ongoing research and presents future prospects for the potential therapeutics of nanoformulations of anticancer and antiangiogenic drug combinations for more effective combination cancer therapy.  Co-assembled nanoparticles provided higher therapeutic efficacy against tumor progression compared with free drug monotherapy or their free combination [81] Polymeric nanoparticles Methoxypoly (ethylene glycol)-block-poly(d,l-lactide) copolymer and poly(acrylic acid-co-4-vubylphenylbornonic acid

Nanoformulations of Anticancer and Antiangiogenesis Drugs for Combination Cancer Therapy
Organic (e.g., polymers, lipids) and inorganic (e.g., mesoporous silica nanoparticles or MSNs, gold) material-based nanoformulations have been extensively developed, investigated, and used for dual-drug combinations. In particular, nanoformulations presented in Table 1, developed to deliver

Nanoformulations of Anticancer and Antiangiogenesis Drugs for Combination Cancer Therapy
Organic (e.g., polymers, lipids) and inorganic (e.g., mesoporous silica nanoparticles or MSNs, gold) material-based nanoformulations have been extensively developed, investigated, and used for dual-drug combinations. In particular, nanoformulations presented in Table 1, developed to deliver antiangiogenic and anticancer drugs in order to increase their therapeutic efficacies, are discussed below for their use in combination cancer therapy. The combination of the drugs used in the below-mentioned nanoformulations have been reported to act in a synergistic manner, where the cumulative effect of the antiangiogenic and anticancer drugs is greater than the sum of the individual effects of each drug.

Polymeric-Based Nanoformulations
Polymers offer superior advantages and vast usage for nanodrug delivery among all the commonly used biodegradable nanomaterials. Biocompatible polymers provide a versatile platform to load multiple low molecular weight drugs to generate different types of nanoformulations, such as polymer nanoparticles, polymer-drug conjugates, polymer-based micelles, dendrimers, and hydrogels. Hence, this section highlights and discusses the recent advances and impacts of polymer-based nanoformulations, which have shown potential effectiveness for combination cancer therapy.
Jinming Zhang et al. developed a pH-sensitive polymeric nanoparticle using an amphiphilic poly (b-amino ester) copolymer to deliver Dox and curcumin (Cur) in optimized ratios for the treatment of hepatocellular carcinoma (HCC) [80]. Dox is commonly used as anticancer drug that causes DNA damage and activate apoptosis. Cur is a bioactive compound derived from the herb Curcuma longa L. (known as turmeric) and exhibits a potent antiangiogenic activity. The Dox and Cur nanoformulation showed a low polydispersity, high encapsulation efficiency, and enhanced release in the acidic environment of tumor cells. Likewise, an enhanced cellular internalization was observed in human liver cancer cells and human umbilical vein endothelial cells as compared to their free-drug counterparts. This nanoformulation also exhibited a high rate of apoptosis in human liver cancer cells and greater antiangiogenic effects both in vitro and in vivo ( Figure 3). Overall, this pH-sensitive polymeric nanocarrier containing Dox and Cur drugs was demonstrated to inhibit cancer cell proliferation and angiogenesis in a synergistic manner, suppressing the tumor growth in HCC [80]. antiangiogenic and anticancer drugs in order to increase their therapeutic efficacies, are discussed below for their use in combination cancer therapy. The combination of the drugs used in the belowmentioned nanoformulations have been reported to act in a synergistic manner, where the cumulative effect of the antiangiogenic and anticancer drugs is greater than the sum of the individual effects of each drug.

Polymeric-Based Nanoformulations
Polymers offer superior advantages and vast usage for nanodrug delivery among all the commonly used biodegradable nanomaterials. Biocompatible polymers provide a versatile platform to load multiple low molecular weight drugs to generate different types of nanoformulations, such as polymer nanoparticles, polymer-drug conjugates, polymer-based micelles, dendrimers, and hydrogels. Hence, this section highlights and discusses the recent advances and impacts of polymerbased nanoformulations, which have shown potential effectiveness for combination cancer therapy.
Jinming Zhang et al. developed a pH-sensitive polymeric nanoparticle using an amphiphilic poly (b-amino ester) copolymer to deliver Dox and curcumin (Cur) in optimized ratios for the treatment of hepatocellular carcinoma (HCC) [80]. Dox is commonly used as anticancer drug that causes DNA damage and activate apoptosis. Cur is a bioactive compound derived from the herb Curcuma longa L. (known as turmeric) and exhibits a potent antiangiogenic activity. The Dox and Cur nanoformulation showed a low polydispersity, high encapsulation efficiency, and enhanced release in the acidic environment of tumor cells. Likewise, an enhanced cellular internalization was observed in human liver cancer cells and human umbilical vein endothelial cells as compared to their free-drug counterparts. This nanoformulation also exhibited a high rate of apoptosis in human liver cancer cells and greater antiangiogenic effects both in vitro and in vivo ( Figure 3). Overall, this pHsensitive polymeric nanocarrier containing Dox and Cur drugs was demonstrated to inhibit cancer cell proliferation and angiogenesis in a synergistic manner, suppressing the tumor growth in HCC [80].  and polyethylene glycol-vitamin E succinate (PEG-VES). These could be directly self-assembled into SCN due to their intermolecular hydrophobic interactions, thereby combining two drugs within a single nanocarrier [81]. These SCNs presented superior effects in vitro (using BEL-7402 and Hep G2 cancer cells) in comparison to Sora, Cur, and their physical mixture (Sora + Cur) in terms of enhanced cytotoxicity, cell apoptosis, and antiangiogenesis activities in tube formation and micro vessel formation from aortic rings. Specifically, in a tumor xenograft model of human hepatocellular carcinoma cell lines of BEL-7402, SCN showed an enhanced inhibitory effect on tumor progression when compared to free drugs or their physical mixture, along with significantly greater antiproliferation and antiangiogenesis properties. Thus, these co-delivered nanoassemblies of Sora and Cur have been shown to enhance the therapeutic effect on antiangiogenesis and antiproliferation activities for combination cancer therapy in an in vivo model of hepatic cellular carcinoma ( Figure 4) [81].
Pharmaceutics 2020, 12, x 9 of 27 In another study, Haiqiang Cao et al. designed polymeric self-assembled nanoparticles (SCNs) with sizes of 84.97 ± 6.03 nm (homogeneous nanometric spherical particles) to enhance the therapeutic effect in HCC. This nanoparticle comprised of two hydrophobic drugs-sorafenib (Sora) and Cur-and polyethylene glycol-vitamin E succinate (PEG-VES). These could be directly selfassembled into SCN due to their intermolecular hydrophobic interactions, thereby combining two drugs within a single nanocarrier [81]. These SCNs presented superior effects in vitro (using BEL-7402 and Hep G2 cancer cells) in comparison to Sora, Cur, and their physical mixture (Sora + Cur) in terms of enhanced cytotoxicity, cell apoptosis, and antiangiogenesis activities in tube formation and micro vessel formation from aortic rings. Specifically, in a tumor xenograft model of human hepatocellular carcinoma cell lines of BEL-7402, SCN showed an enhanced inhibitory effect on tumor progression when compared to free drugs or their physical mixture, along with significantly greater antiproliferation and antiangiogenesis properties. Thus, these co-delivered nanoassemblies of Sora and Cur have been shown to enhance the therapeutic effect on antiangiogenesis and antiproliferation activities for combination cancer therapy in an in vivo model of hepatic cellular carcinoma ( Figure 4) [81].  in an in vivo study indicated that the CA4/SN38 co-encapsulated polymeric nanoparticles displayed synergistic activities in inhibiting the tumor growth ( Figure 5) [82].
Wen Jing Yang et al. attempted using poly(acrylic acid-co-4-vinylphenylboronic acid) to develop a synergistic pH/redox stimuli-responsive nanohydrogel for the sequential local delivery of combretastatin A-4 phosphate (CA4P) and Dox for antiangiogenesis and anticancer combination therapy. This nanoformulation also released CA4P and Dox in a sequential manner at the target site on demand. A cumulative release was observed, with about 57.2% released at 9 h and almost 90.7% after 48 h at pH 6.5. This nanogel exhibited a high inhibitory activity on the cancer cell proliferation (MCF-7 and normal 3T3-L1 cells) in vitro, with a superior antitumor therapeutic efficacy with a single injection in HCC xenograft tumor-bearing mice ( Figure 6) [83].  [82]. Reprinted with permission from ref. [82], copyright (2017) American Chemical Society. * p < 0.05, *** p < 0.001.
Wen Jing Yang et al. attempted using poly(acrylic acid-co-4-vinylphenylboronic acid) to develop a synergistic pH/redox stimuli-responsive nanohydrogel for the sequential local delivery of combretastatin A-4 phosphate (CA4P) and Dox for antiangiogenesis and anticancer combination therapy. This nanoformulation also released CA4P and Dox in a sequential manner at the target site on demand. A cumulative release was observed, with about 57.2% released at 9 h and almost 90.7% after 48 h at pH 6.5. This nanogel exhibited a high inhibitory activity on the cancer cell proliferation (MCF-7 and normal 3T3-L1 cells) in vitro, with a superior antitumor therapeutic efficacy with a single injection in HCC xenograft tumor-bearing mice ( Figure 6) [83]. Mohyeddin Assali et al. prepared self-assembled micelles combining combretastatin A4 (CA4) and camptothecin (CPT) by using click chemistry. These micelles displayed enhanced stability and water solubility at pH 7.4, with a low critical micelle concentration (CMC) of 0.9 mM. Furthermore, this micelle formulation encompassing two drugs displayed five times higher cytotoxicity against HeLa cancer cells when compared to the free drugs. Moreover, a combination index (CI) of less than 1 suggested a synergistic activity by the micelles. Imaging studies of HeLa cells treated with FITCloaded micelles showed a rapid internalization. Based on these results, in vivo studies to determine the anticancer activity were suggested [84].
Fatima Zohra Dahmani et al. developed a heparin-gambogic acid conjugate (cRHG) and c(RGDyK)-functionalized (targeting ligand) self-assembled polymeric amphiphilic Mohyeddin Assali et al. prepared self-assembled micelles combining combretastatin A4 (CA4) and camptothecin (CPT) by using click chemistry. These micelles displayed enhanced stability and water solubility at pH 7.4, with a low critical micelle concentration (CMC) of 0.9 mM. Furthermore, this micelle formulation encompassing two drugs displayed five times higher cytotoxicity against HeLa cancer cells when compared to the free drugs. Moreover, a combination index (CI) of less than 1 suggested a synergistic activity by the micelles. Imaging studies of HeLa cells treated with FITC-loaded micelles showed a rapid internalization. Based on these results, in vivo studies to determine the anticancer activity were suggested [84].
Fatima Zohra Dahmani et al. developed a heparin-gambogic acid conjugate (cRHG) and c(RGDyK)-functionalized (targeting ligand) self-assembled polymeric amphiphilic nanoformulations. These nanoformulations showed considerable the inhibition of VEGF, hypoxia inducible factor-1 alpha, and CD31 expression, with the significant downregulation of phosphorylated vascular endothelial growth factor receptor-2 (pVEGFR2). These results also demonstrated a versatile nanoplatform for efficient combinational tumor therapy in vivo (Figure 7). Hence, a combination of chemo-and antiangiogenesis therapy holds immense potential for effective tumor growth inhibition [85].
Pharmaceutics 2020, 12, x 12 of 27 nanoformulations. These nanoformulations showed considerable the inhibition of VEGF, hypoxia inducible factor-1 alpha, and CD31 expression, with the significant downregulation of phosphorylated vascular endothelial growth factor receptor-2 (pVEGFR2). These results also demonstrated a versatile nanoplatform for efficient combinational tumor therapy in vivo ( Figure 7). Hence, a combination of chemo-and antiangiogenesis therapy holds immense potential for effective tumor growth inhibition [85].
C D shows that PTX and ALN were conjugated to HPMA co-polymer via a phe-lys-p-aminobenzyl carbonate (FK-PABC), which showed a sustained release by cathepsin B. This conjugate showed the highest antitumor activity compared to free PTX or with a combination of free PTX plus ALN in 4T1-mcherry mammary adenocarcinoma in the tibia (Figure 8) [86].  Figure 8A) shows that PTX and ALN were conjugated to HPMA co-polymer via a phe-lysp-aminobenzyl carbonate (FK-PABC), which showed a sustained release by cathepsin B. This conjugate showed the highest antitumor activity compared to free PTX or with a combination of free PTX plus ALN in 4T1-mcherry mammary adenocarcinoma in the tibia (Figure 8) [86].
(A)  samples collected on day 11. Data represent mean (SEM of six mice per group. * P < 0.05 value of mice treated with HPMA copolymer-PTX-ALN conjugate was analyzed against saline control mice. * P < 0.05 value of free PTX or combination of free PTX plus ALN was analyzed against control mice treated with PTX vehicle. [86]. Reprinted with permission from ref. [86], copyright (2011) American Chemical Society.
Deepa A Rao et al. developed individual and mixed micelles of polymeric-paclitaxel (PTX) conjugates and polymeric-rapamycin (RAP) conjugates with an acid-sensitive linker. This acidsensitive linker released the drugs from the micelles in a preferential manner at pH 5.5 in lysosomes. The micelles displayed antiproliferative activity in a synergistic manner against ovarian cell lines (human Caucasian ovarian adenocarcinoma and human ovarian clear cell carcinoma) and endothelial cell line. The inhibition of endothelial cell migration and tube formation were also reported. No acute toxicity was observed in healthy mice for over 21 days at a dose of 60 mg/kg of the micelles. The individual micelles exhibited only antiangiogenic activity, whereas the mixed micelles demonstrated both antiangiogenic and apoptosis induction activity in an epithelial ovarian cancer xenograft model in efficacy studies at 20 mg/kg/drug dosed every 4 days and assessed after 21 days (Figure 9) [87].  Here, Dox and CA4 were encapsulated into micelles (namely, RFPMs-DOX-CA4) of 29.2 ± 2.5 nm size. These micelles (RFPMs-DOX-CA4) was demonstrated to sequentially release both drugs, resulting in the sequential killing of endothelial cells and tumor cells in vitro. In addition, these micelles were reported to exhibit a greater tumor growth inhibition and higher survival rate in comparison to free drugs and their combination in B16-F10 tumor-bearing mice. Further, the in vivo evaluation of these micelles showed a significant reduction in the tumor vasculature and cell proliferation, which suggests these micelles can be effective for combination cancer therapy ( Figure 10) [88]. micelles of poly(ethylene glycol)-b-poly(D,L-lactide) (PEG-PLA). Here, Dox and CA4 were encapsulated into micelles (namely, RFPMs-DOX-CA4) of 29.2 ± 2.5 nm size. These micelles (RFPMs-DOX-CA4) was demonstrated to sequentially release both drugs, resulting in the sequential killing of endothelial cells and tumor cells in vitro. In addition, these micelles were reported to exhibit a greater tumor growth inhibition and higher survival rate in comparison to free drugs and their combination in B16-F10 tumor-bearing mice. Further, the in vivo evaluation of these micelles showed a significant reduction in the tumor vasculature and cell proliferation, which suggests these micelles can be effective for combination cancer therapy ( Figure 10) [88].  [88]. Reprinted with permission from ref. [88], copyright (2011) Elsevier. **p < 0.01.
In summary, this section explored cancer targeting through polymer-based nanoformulations of anticancer and antiangiogenic agents in a single nanocarrier for combination therapy at lower doses, with an aim to reduce the toxicity and enhance the therapeutic efficacy.
The hallmark of these above-mentioned nanoformulations is the selection of appropriate polymers, which could be based on polymeric nanoparticles, polymeric conjugates, micelles, and hydrogels. The ideal polymer would be one which is non-toxic, water soluble, non-immunogenic, and has a high drug loading capacity. Apart from this, one of the key factors that plays a critical role in polymer pharmacokinetics and biodistribution is the molecular weight (MW) of the polymer, which ensures a long circulation in the bloodstream to allow EPR-mediated accumulation. Indeed, for non-biodegradable polymers, the MW must be less than 40-50 kDa (renal clearance threshold) to ensure renal elimination [95]. In general, a MW of 30-100 kDa is employed as an optimum range for drug delivery; however, it needs to be tailored to the particular polymer based on its architecture and biodegradability. Considering the key attributes of the polymers, as stated above, significant efforts were made to elucidate the synergetic effects achieved by the combination therapy, which led to advanced drug delivery strategies rather than simply an additive effect of the partner drugs [96,97]. For instance, D-a-tocopheryl poly-ethylene glycol-block-poly (b-amino ester) amphiphilic copolymers used a pH-sensitive nanoparticle with an enhanced pH sensitivity and stability in the physiological environment and encapsulated anticancer and antiangiogenic drugs simultaneously by self-assembly [80]. In another study, a PEG derivative of vitamin E succinate and CA4 and SN38 derivatives could be directly self-assembled into polymeric nanoparticles due to the intermolecular hydrophobic interactions among them, which combined two drugs within a nanovehicle to exert the  [88]. Reprinted with permission from ref. [88], copyright (2011) Elsevier. ** p < 0.01.
In summary, this section explored cancer targeting through polymer-based nanoformulations of anticancer and antiangiogenic agents in a single nanocarrier for combination therapy at lower doses, with an aim to reduce the toxicity and enhance the therapeutic efficacy.
The hallmark of these above-mentioned nanoformulations is the selection of appropriate polymers, which could be based on polymeric nanoparticles, polymeric conjugates, micelles, and hydrogels. The ideal polymer would be one which is non-toxic, water soluble, non-immunogenic, and has a high drug loading capacity. Apart from this, one of the key factors that plays a critical role in polymer pharmacokinetics and biodistribution is the molecular weight (MW) of the polymer, which ensures a long circulation in the bloodstream to allow EPR-mediated accumulation. Indeed, for non-biodegradable polymers, the MW must be less than 40-50 kDa (renal clearance threshold) to ensure renal elimination [95]. In general, a MW of 30-100 kDa is employed as an optimum range for drug delivery; however, it needs to be tailored to the particular polymer based on its architecture and biodegradability. Considering the key attributes of the polymers, as stated above, significant efforts were made to elucidate the synergetic effects achieved by the combination therapy, which led to advanced drug delivery strategies rather than simply an additive effect of the partner drugs [96,97].
For instance, D-a-tocopheryl poly-ethylene glycol-block-poly (b-amino ester) amphiphilic copolymers used a pH-sensitive nanoparticle with an enhanced pH sensitivity and stability in the physiological environment and encapsulated anticancer and antiangiogenic drugs simultaneously by self-assembly [80]. In another study, a PEG derivative of vitamin E succinate and CA4 and SN38 derivatives could be directly self-assembled into polymeric nanoparticles due to the intermolecular hydrophobic interactions among them, which combined two drugs within a nanovehicle to exert the desired effect for effective combination therapy [81]. In another experiment, polymer-based hydrogel was used to deliver antiangiogenic and anticancer drugs with sequential drug release profiles, which subsequently led to the sustained long-term release of the drugs [83]. In one study, HPMA copolymer facilitated the attachment of chemotherapeutic and antiangiogenic drugs to a polymeric backbone, as well as targeting moieties, such as the bone-targeting agent ALN [86]. Overall, these polymer-based nanoformulations help with implementing a robust and successful combinational cancer therapy.

Lipid Based Nanoformulations
Lipid-based nanocarriers, such as liposomes, solid lipid nanoparticles, nanocells, and lipid-coated nanoparticles have emerged as promising nanovehicles for cancer therapy. This section focusses on the recent advancements of lipid-based and other organic nanoformulations for combination chemo-and antiangiogenesis therapy. Shiladitya Sengupta et al. incorporated two drugs (Dox and CA4) for a lipid-based nanoformulation in a two-step process. CA4 was incorporated into the lipid layer, whereas Dox was loaded into the polymeric core. The nanoformulation was preferentially taken up by the tumor within 5 h and inhibited the temporal targeting of tumor cells and neovasculature together in a sequential manner and retained for at least 24 h. The synergistic effects of inhibiting the tumor vessels and proliferation of tumor cells were achieved in in vitro and in vivo studies [89].
In another study, Wenbing Dai et al. constructed a novel liposomal delivery system with the traditional chemotherapy drug Dox and antiangiogenesis agent CA4 with surface modification by the targeting ligand octreotide (Oct). The release kinetics of drugs from the nanoformulation confirmed the rapid release of CA4, followed by a slow release of Dox in vitro. In addition, sequential killing effects were confirmed in vivo using nude mice bearing MCF-7 tumors. The active targeted liposomes Oct-liposome of CA4 and Dox showed a specific cellular uptake through ligand-receptor interaction and a higher antitumor effect in vitro against somatostatin receptor (SSTR) positive cell lines [98][99][100]. This study concluded that the liposome-based nanoformulation can be a potential nanodrug delivery system for the treatment of malignant solid tumors [90].
Yi-Fei Zhang et al. developed an arginine-glycine-aspartic acid (RGD)-modified liposome, co-encapsulating Dox and CA4 with the aim of assessing sequential release and enhancing tumor inhibition responses. The results showed that the release rate of Dox was much slower than that of CA4 in vitro. The intracellular uptake of liposomal drugs by B16/B16F10 melanoma tumor cells and human umbilical vein endothelial cells (HUVECs) was enhanced based on flow cytometry and laser confocal scanning microscopy assessments. A cytotoxicity assay demonstrated the lower half maximal inhibitory concentration (IC 50 ) of RGD-modified liposomes than the corresponding unmodified liposomes. Prominent synergistic effects on tumor reduction were observed with RGD-modified liposomes co-encapsulating CA4 and Dox, delineating the importance of a targeted drug delivery system for the co-encapsulation of antiangiogenic and anticancer agents for cancer treatment (Figure 11) [91].
In summary, the development of lipid-based nanoparticles/nanocarriers, and particularly liposomes, as discussed in the above-cited studies, has been reported as an alternative modality for cancer therapy. These lipid-based nanoformulations work by means of passive and active targeting, thereby reducing the toxicity associated with anticancer and antiangiogenic agents and subsequently improving the efficacy of these drugs similarly to various other types of nanoformulations. Nonetheless, lipid-based nanoparticles have been found to be more beneficial than some of their counterparts, owing to their ingredients being more biocompatible and biodegradable in nature. Moreover, the amphiphilic properties of liposomes allow these nanoparticles to encapsulate both hydrophobic and hydrophilic anticancer and antiangiogenic drugs. Hence, lipid-based nanoparticles may help in improving the therapeutic efficacy and safety profile of combination chemotherapy and, ultimately, the prognosis of cancer patients. In summary, the development of lipid-based nanoparticles/nanocarriers, and particularly liposomes, as discussed in the above-cited studies, has been reported as an alternative modality for cancer therapy. These lipid-based nanoformulations work by means of passive and active targeting, thereby reducing the toxicity associated with anticancer and antiangiogenic agents and subsequently improving the efficacy of these drugs similarly to various other types of nanoformulations. Nonetheless, lipid-based nanoparticles have been found to be more beneficial than some of their counterparts, owing to their ingredients being more biocompatible and biodegradable in nature. Moreover, the amphiphilic properties of liposomes allow these nanoparticles to encapsulate both hydrophobic and hydrophilic anticancer and antiangiogenic drugs. Hence, lipid-based nanoparticles may help in improving the therapeutic efficacy and safety profile of combination chemotherapy and, ultimately, the prognosis of cancer patients.

Inorganic Material-Based Nanoformulations
As evident from the above-mentioned numerous nanoformulations, most nanodrug delivery systems in clinical trials and in clinical use are based on organic (mostly liposomal or polymer) platforms. In particular, liposomal formulations-e.g., Doxil-used for monotherapy for cancer as well in combination chemotherapy have been successfully translated to the clinic. Meanwhile, nanoformulations based on inorganic materials such as MSNs, iron oxides (Fe3O4), gold (Au), and silver (Ag) have also become a very important class of nanodrug delivery vehicle. The following discussion illustrates the recent advances in inorganic-based nanoformulations, especially MSNs, iron oxide, and gold-based nanoformulations, for chemo and antiangiogenesis combination cancer therapy.  [91]. Reprinted with permission from ref. [91], copyright (2010) Elsevier.

Inorganic Material-Based Nanoformulations
As evident from the above-mentioned numerous nanoformulations, most nanodrug delivery systems in clinical trials and in clinical use are based on organic (mostly liposomal or polymer) platforms. In particular, liposomal formulations-e.g., Doxil-used for monotherapy for cancer as well in combination chemotherapy have been successfully translated to the clinic. Meanwhile, nanoformulations based on inorganic materials such as MSNs, iron oxides (Fe 3 O 4 ), gold (Au), and silver (Ag) have also become a very important class of nanodrug delivery vehicle. The following discussion illustrates the recent advances in inorganic-based nanoformulations, especially MSNs, iron oxide, and gold-based nanoformulations, for chemo and antiangiogenesis combination cancer therapy.
Sunhui Chen et al. developed bovine serum albumin-coated superparamagnetic iron oxide (BSA-SPIO) nanoparticles and further co-loaded this nanosystem into two drugs-the cytotoxic drug Cur and tyrosine kinase inhibitor (TKI) sunitinib (Sun)-to achieve synergistic effect. The BSA-SPIOs and dual-drug (Sun and Cur)-loaded BSA-SPIO nanoparticles, when compared to free drugs, displayed the most significant tumor inhibition in an MCF-7 tumor xenograft mouse model. In addition, these NPs were used for in vivo MR imaging and prompted a good targeting to the tumor site with well-preserved stability and long-circulation potential, rendering them promising candidates for both tumor diagnosis and therapy. These nanoparticles exhibit both in vivo MR imaging and tumor therapy capability, thus being a promising theragnostic material ( Figure 12) [92]. and dual-drug (Sun and Cur)-loaded BSA-SPIO nanoparticles, when compared to free drugs, displayed the most significant tumor inhibition in an MCF-7 tumor xenograft mouse model. In addition, these NPs were used for in vivo MR imaging and prompted a good targeting to the tumor site with well-preserved stability and long-circulation potential, rendering them promising candidates for both tumor diagnosis and therapy. These nanoparticles exhibit both in vivo MR imaging and tumor therapy capability, thus being a promising theragnostic material ( Figure 12) [92]. Dox. Additionally, 9-amino acid (CRGDKGPDC) cyclic (iRGD) peptide was used as a targeting ligand conjugated onto the MSN surface. Particularly, iRGD peptide targeted α 2 β 3 integrin receptors, which are overexpressed in cancer and tumor vascular cells. Therefore, this nanocarrier targeted tumor vasculature specifically and was used for combined chemo-and antiangiogenesis therapy. When these dual-drug-loaded MSNs were injected into the blood circulation, they accumulated at the targeted tumor via the α 2 β 3 integrin receptors in the tumor environment. Most of the antiangiogenic drug was released first, while only a small amount of Dox was released during the same time period due to the negatively charged MSNs interacting electrostatically and via hydrogen bonding with the positively charged Dox. Sequentially, after reaching deep into the tumor, Dox quickly released in the lower pH environment. The further uptake by tumor cells and release of Dox efficiently induced the apoptosis of the cancer cells. Such nanoformulation showed a synergetic effect and greatly enhanced the cytotoxic effect of Dox in cancer cells ( Figure 13) [93].
which are overexpressed in cancer and tumor vascular cells. Therefore, this nanocarrier targeted tumor vasculature specifically and was used for combined chemo-and antiangiogenesis therapy. When these dual-drug-loaded MSNs were injected into the blood circulation, they accumulated at the targeted tumor via the α2 β3 integrin receptors in the tumor environment. Most of the antiangiogenic drug was released first, while only a small amount of Dox was released during the same time period due to the negatively charged MSNs interacting electrostatically and via hydrogen bonding with the positively charged Dox. Sequentially, after reaching deep into the tumor, Dox quickly released in the lower pH environment. The further uptake by tumor cells and release of Dox efficiently induced the apoptosis of the cancer cells. Such nanoformulation showed a synergetic effect and greatly enhanced the cytotoxic effect of Dox in cancer cells ( Figure 13) [93]. Step 1: CA4 is first released at tumor vasculature with the help of targeting ligands (iRGD peptides); subsequently, in step 2 this nanoformulation is endocytosed into acidic tumor cells where most of the Dox is released [93]. Reprinted with permission from ref. [93], copyright (2016) Dove press. You-Hong You et al. engineered a multifunctional (PDA)-coated gold (Au) nanostar (NS@PPFA) nanoformulation containing Dox (NS-D@PPFA) for combination cancer therapy to target drug resistance in breast cancer. Breast cancer MCF-7 and drug-resistant MCF-7/Adriamycin (ADR) cells demonstrated the effective intracellular uptake and cytotoxicity of the designed nanoformulation. These nanoagents were found to be more active in inhibiting VEGF-induced VEGFR angiogenesis. The CD31 and pVEGFR2 levels were also significantly reduced in vivo. The investigation of the antitumor activity of NS-D@PPFA (6 mg/kg Au, 1.8 mg/kg Dox) in comparison to PBS as a control and free Dox (5 mg/kg), plus near-infrared (NIR) laser in MCF-7/ADR tumor-bearing mice, showed that the inhibition of the tumor cells and endothelial cells proliferation was achieved by combined chemo and photothermal effects (chemo-PTT). The photothermal effect and triggered drug release was induced by NIR laser (808 nm)/808-nm irradiation ( Figure 14) [94]. From the in vitro and in vivo results, this nanoformulation simultaneously presented a remarkable antitumor efficacy by chemo-PTT combination therapy, triggered by a single NIR laser ( Figure 14) [94]. Overall, this study found to a new therapeutic strategy against antiangiogenic cancer therapy and multidrug-resistant cancers. Step 1: CA4 is first released at tumor vasculature with the help of targeting ligands (iRGD peptides); subsequently, in step 2 this nanoformulation is endocytosed into acidic tumor cells where most of the Dox is released [93]. Reprinted with permission from ref. [93], copyright (2016) Dove press. You-Hong You et al. engineered a multifunctional (PDA)-coated gold (Au) nanostar (NS@PPFA) nanoformulation containing Dox (NS-D@PPFA) for combination cancer therapy to target drug resistance in breast cancer. Breast cancer MCF-7 and drug-resistant MCF-7/Adriamycin (ADR) cells demonstrated the effective intracellular uptake and cytotoxicity of the designed nanoformulation. These nanoagents were found to be more active in inhibiting VEGF-induced VEGFR angiogenesis. The CD31 and pVEGFR2 levels were also significantly reduced in vivo. The investigation of the antitumor activity of NS-D@PPFA (6 mg/kg Au, 1.8 mg/kg Dox) in comparison to PBS as a control and free Dox (5 mg/kg), plus near-infrared (NIR) laser in MCF-7/ADR tumor-bearing mice, showed that the inhibition of the tumor cells and endothelial cells proliferation was achieved by combined chemo and photothermal effects (chemo-PTT). The photothermal effect and triggered drug release was induced by NIR laser (808 nm)/808-nm irradiation ( Figure 14) [94]. From the in vitro and in vivo results, this nanoformulation simultaneously presented a remarkable antitumor efficacy by chemo-PTT combination therapy, triggered by a single NIR laser ( Figure 14) [94]. Overall, this study found to a new therapeutic strategy against antiangiogenic cancer therapy and multidrug-resistant cancers.
In summary, given the variety of inherent properties that different inorganic materials possess-as discussed above, for instance superparamagnetic (iron oxides) and photothermal (gold) activity-these are highly interesting constructs to be included in the design of hybrid nanocarriers for combination therapies. Many inorganic materials possess inherent imaging activity, rendering them suitable to be tracked by different biological or medical imaging techniques. As mentioned above, this not only allows easy tracking in the biological or physiological environment during the investigation of the behavior of the nanocarrier, but also implies their potential as theragnostic agents. A couple of other examples showed how the modularity and robustness of the mesoporous silica matrix could be utilized to load multiple drugs simultaneously, while separately functionalizing the outside particle surface with targeting ligands. Nevertheless, inorganic materials usually require organic functionalization to reach the desired responsiveness and, in many cases, biocompatibility in the physiological environment, as well as for the conjugation of targeting ligands. In the above-discussed cases, organic coatings were utilized, e.g., to facilitate pH-sensitive drug release (PDA) and long circulation time, and low immunogenicity (BSA) and drug loading capability. Such properties are usually dependent on the interaction of the nanosystem with the surrounding environment (sometimes referred to as the "bio-nano interface"), while the inherent properties of inorganic materials (robustness for drug incorporation and protection, imaging activity, photothermal and photodynamic activity) are not in general dependent on direct contact. Consequently, constructing hybrid nanocarriers by making use of an inorganic platform with organic functionalization appears to be an especially flexible approach for the development of synergistic nanocarriers for combined chemo-and antiangiogenetic therapy, even combined with other therapeutic strategies. In summary, given the variety of inherent properties that different inorganic materials possessas discussed above, for instance superparamagnetic (iron oxides) and photothermal (gold) activitythese are highly interesting constructs to be included in the design of hybrid nanocarriers for combination therapies. Many inorganic materials possess inherent imaging activity, rendering them suitable to be tracked by different biological or medical imaging techniques. As mentioned above, this not only allows easy tracking in the biological or physiological environment during the investigation of the behavior of the nanocarrier, but also implies their potential as theragnostic agents. Tumor growth curve of mice receiving intravenously administration of PBS, Dox (5 mg/kg), and NS-D@PPFA (6 mg/kg Au; 1.8 mg/kg Dox) at day 0 and day 14, respectively. Tumors were treated with NIR irradiation (0.9 W/cm 2 , 3 min, 3 times) at 24 h and 48 h after each injection. (B) Tumor weight was measured on excised tumors at day 28 after different treatments. (C) Body weight monitoring of the treated mice over a period of 28 days [94]. Reprinted with permission from ref. [94]. * p < 0.05, N.S.: not significant.

Future Perspectives, Outlook, and Conclusions
On one hand, the drug resistance and multiple side effects associated with conventional chemotherapy remains a significant barrier in the treatment of cancer. On the other hand, monotherapy of antiangiogenic drugs has demonstrated only a temporary response in clinical trials, with some serious dose limiting toxicities and allergic reactions. Nevertheless, the tumor tissue exposure to combination therapy of antiangiogenesis and chemotherapeutic agents with dissimilar chemical and pharmacological properties remains very challenging. To overcome this, various nanoformulations with tunable and predictable release of multiple drugs have been investigated in preclinical studies (mice models), and the same needs to be validated in different species such as rat, rabbit, and monkey and thus eventually among cancer patients before these nanoformulations can be launched into the market.
Despite the wide array of advantages of nanoformulation-mediated combination chemo-antiangiogenesis cancer therapy-e.g., reduction in tumor growth, the regulation of drug resistance, and overall synergistically improved therapeutic efficacy-there are certain challenges that remain in their preparation and efficient translation. These include unknown toxicity and immunogenicity aspects, inefficient drug targeting, the lack of collaboration from bench to bedside between experts in the particular fields, and the cost of industrial production. Although nanotechnology has had a major contribution in medicine, particularly in cancer therapy, a common nanotoxicology consensus remains undefined [50]. For instance, long-term toxicity and safety profiles associated with nanoparticles need to be determined, but so far acute toxicity have largely been explored on a case-by-case basis. Specifically, the molecular interactions between nanocarriers and the endothelial cell lining should be unraveled, as most of these drug formulations are administered intravenously. In order to minimize the systemic toxicity, certain design principles can be considered, such as overall size and physical and chemical surface properties, as well as the selection and tailoring of the nanoformulations [101]. In the best-case scenario, functionalizing the nanocarrier with targeting ligands should help to reduce side effects by selectively increasing the drug accumulation at target sites and thereby reducing systemic exposure, eventually improving the therapeutic outcome [102]. Additionally, novel screening tests need to be designed to assess the biodistribution and understand the biochemical pathways that regulate cell functions. Although liposome-based nanoformulations are frequently used due to their non-immunogenicity, their short half-life, low solubility, and high production costs are to their disadvantage [103]. Polymeric-based nanoformulations have the advantage of being biodegradable, but at the same time, their drug release takes place in an uncontrolled manner, which is disadvantageous from an optimal nanoformulation point of view [104,105]. Moreover, there is a need to consider the cost of production for these nanoformulations for clinical use, as the cost of these combined chemo-antiangiogenic nanoformulations may supposedly be higher than the total cost of each drug altogether. The potential solution to the above-mentioned challenges lies in bringing the interdisciplinary experts and collaborators from academics, clinicians, scientists, and regulatory bodies to ensure a goal to establish the improved therapeutic outcomes of combined chemo-antiangiogenesis therapy and, ultimately, improving the quality of life of cancer patients.
Furthermore, the exploration of nanoformulations comprising anticancer drugs in combination with anti-inflammatory drugs [106][107][108], radioligands [109][110][111][112] and specific target genes is highly warranted [113]. These approaches would be beneficial in improving the therapeutic outcome, in addition to studies addressing the long-term toxicity and safety of the nanoformulations. Different biodegradable linkers can also be engineered to deliver the parent drugs through a controllable and targetable fashion, with the drug released sequentially or simultaneously via specific mechanisms in the tumor microenvironment, with the potential to achieve great advances in cancer therapy. Moreover, the incorporation of imaging agents (e.g., radionuclides or inorganic constructs) would also allow clinicians to tailor combination therapies in a more personalized manner and/or for complex cancers such ovarian cancer [114,115]. In conclusion, nanoformulations based on different nanocarriers, such as polymeric nanoparticles, micelles, hydrogels, liposomes, mesoporous silica nanoparticles, and gold nanoparticles containing anticancer and antiangiogenic agents have been established as a promising cancer treatment strategy in preclinical models. Nevertheless, there is still a long road for clinical application in terms of patient outcomes; however, these approaches offer flexible and robust platforms for the realization of the presented combination therapy concepts down the road.