Emerging concepts in designing next-generation multifunctional nanomedicine for cancer treatment

Abstract Nanotherapy has emerged as an improved anticancer therapeutic strategy to circumvent the harmful side effects of chemotherapy. It has been proven to be beneficial to offer multiple advantages, including their capacity to carry different therapeutic agents, longer circulation time and increased therapeutic index with reduced toxicity. Over time, nanotherapy evolved in terms of their designing strategies like geometry, size, composition or chemistry to circumvent the biological barriers. Multifunctional nanoscale materials are widely used as molecular transporter for delivering therapeutics and imaging agents. Nanomedicine involving multi-component chemotherapeutic drug-based combination therapy has been found to be an improved promising approach to increase the efficacy of cancer treatment. Next-generation nanomedicine has also utilized and combined immunotherapy to increase its therapeutic efficacy. It helps in targeting tumor immune response sparing the healthy systemic immune function. In this review, we have summarized the progress of nanotechnology in terms of nanoparticle designing and targeting cancer. We have also discussed its further applications in combination therapy and cancer immunotherapy. Integrating patient-specific proteomics and biomarker based information and harnessing clinically safe nanotechnology, the development of precision nanomedicine could revolutionize the effective cancer therapy.


Introduction
Cancer remains the most fatal and life-threatening disease worldwide, causing an estimated 19.3 million new cases and 10.0 million deaths in 2020 [1]. Current therapeutic approaches are not found to completely cure the advanced cancer having distant organ metastases. Treatment of cancer follows several approaches, including locoregional (surgery and radiation therapy), chemotherapy, endocrine (hormone) therapy, targeted therapy, etc. Such therapeutic approaches also possess certain disadvantages such as fatigue, numbness, nail changes, hair loss, loss of appetite, mouth sores, nausea, weight changes, vomiting, diarrhoea, heart damage, etc [2][3][4][5][6]. In spite of its huge potential, chemotherapy remains disadvantageous in having off-target side effects and non-specific delivery [7]. Nanotechnology has become a promising approach to overcome this limitation. The nano-sized materials provide opportunities for their use in diagnosing, monitoring, controlling, preventing and treating diseases [8]. The concept of nanoparticles was first adapted by Nobel laureate Richard P. Feynman in his famous lecture entitled "There's plenty of room at the bottom" in December 1959 [9]. Nanotherapy is the therapeutic strategy harboring nanoparticles ranging from 10 to 100 nm for intravenous delivery [10,11]. Nanoparticles below 10 nm diameter are prone to renal clearance [11]. Widely used examples of nanoparticles having anticancer efficacy are Doxil, a liposomal formulation of Doxorubicin and Abraxane, an albumin-bound nanoparticle of paclitaxel [12]. These drugs also received approval from U.S. Food and Drug Administration (FDA). Apart from that, several other nanotechnology platforms like organic, inorganic and organometallic nanoparticles have also been used over the past two decades for therapeutic, diagnostic and theranostic purposes [13]. Nanoparticles can be easily custom-tailored and advantageous in having the following features: (1) carry a high payload of biologically active drug [11,14,15], (2) protects the drug from degradation, (3) contain large surface area to accommodate multiple targeting ligands (4) larger surface to interact with multiple types of drug molecules, (5) controlled release profile of drug and (6) potential to bypass multidrug resistant mechanism [11,16,17].
Nanotechnology also undergoes several barriers that hinder its successful translation into the clinic. Various biological barriers limit the functionality of the nanoparticles and their clinical outcome. Nanoparticles get easily phagocytosed and degraded by the macrophages of the liver, spleen, lungs, lymph nodes and skin [13]. According to recent statistics, 0.7% of the total injected dose can get targeted to tumor. The primary reason for this failure is the formation of protein corona (proteins adsorbed on the nanoparticle from plasma and/or intracellular fluid) around nanoparticles and their subsequent opsonization (the process where opsonins or extracellular proteins bind to the surface of the nanoparticles, causing the degradation of nanoparticles by phagocytes) and eventual phagocytosis [10,13]. High encapsulation efficiency of a drug does not always lead to high therapeutic potential. Again, hydrophobic drugs which precipitate over time also do not show significant efficacy [10]. Therefore, designing a potent yet stable nanoparticle has always been challenging. In the following section, we will focus on the strategies acquired by the researchers in designing effective nanoparticles to aid anticancer therapy and we will also describe the progress made so far with nanotherapy.
By designing suitable combinatorial nanotherapeutics, one can achieve targeted delivery, reduce side effects of free drugs, delay in developing drug resistance and accomplish synergistic drug interactions at low doses [18]. Oncogenic signaling pathways can also be targeted using nanoparticles to selectively target the tumor cells without causing systemic toxicity. Moreover, nanomedicine mediated anti-angiogenic drug delivery makes them more reachable towards tumor vasculature. Furthermore, nanoparticles potentiate the gene therapy to introduce therapeutic nucleic acids into target cells to achieve curative response in cancer patients [19]. Recently, nanotherapy has also been used to deliver immunotherapeutic drugs in combination with conventional therapeutic modalities (e.g., chemotherapy, RNAi therapy, photothermal, photodynamic and radiotherapy) to increase anti-tumor efficacy [20]. Minimally invasive photothermal therapy allows killing of cancer cells by heat generated upon their exposure to the near infrared (NIR) light [20]. Similarly, photodynamic therapy utilizes photosensitizers which upon exposure to light releases reactive oxygen species and induce cellular toxicity toward tumors [20]. Radiotherapy induced radiation damage of cancer cells is widely used in cancer treatment, which allows curative treatment of 40% patients out of >50% of the patients with cancer treated by radiotherapy [20,21]. Cancer immunotherapy has gained tremendous attention for providing long-term treatment in cancer, as it facilitates immunological memory induced delay in cancer remission. One of the key challenges of implementing immunotherapy is off-target responses. Nanotherapy has potential to overcome this challenge by delivering these immunotherapeutic drugs to desired target sites [22]. In this review, we have discussed the potential applications and the strength of nanotherapy by combining therapies like chemotherapy, gene therapy and immunotherapy. The central theme of this review is on engineering multifunctional cancer nanotherapy as described in Figure 1.

Nanoparticle designing
Engineering multifunctional nanoparticle or precision nanomedicine still remains challenging. The primary aim of nanoparticle design is to transport therapeutic drugs and imaging agents. Such cargos are loaded on to nanoparticles either by chemical conjugation or by encapsulation [7]. Researchers found that the encapsulation technique remains more effective for clinical translation of a low potency drug than chemical conjugation. In order to produce efficacy, the drug should comprise at least 10% (wt/wt) of the entire nanoparticle composition [13]. While designing nanoparticles, certain criteria are needed to be taken into consideration, such as stability, pharmacological reasonability, pathophysiological suitability, etc [13]. Current development of cancer nanomedicine has provided answers to problems like (1) prolonged blood circulation to improve stability and bioavailability, (2) adequate tumor accumulation and (3) controlled drug release and uptake by tumor cells having a release profile for specific targeting [16,17].

Prolonged blood circulation to improve bioavailability
Proteins present in biological fluids get adsorbed in the nanoparticles when administered. Such nanoparticle-protein complexes get easily recognized by macrophages and phagocytosed [10,23,24]. To overcome this difficulty, poly(ethylene glycol) (PEG) is often used to form a hydrophilic "stealth" [25]. PEGylation promotes solubilization of the nanoparticles, prevents opsonization and increases the half-life of the drug in the blood stream [26]. Doxil (liposomal doxorubicin) is a commonly used PEGylated nanoparticle. It shows ∼100 times longer half-life compared with free doxorubicin in circulation [27]. Current research has utilized nanotechnology to increase the solubility of potent but poorly soluble drugs such as cyclosporine, paclitaxel, amphotericin B, etc [28]. , thiol-thiol conjugation (cleaved under reducing condition), gold-thiol conjugation (stable), thiol/maleimide conjugation (stable), azide/alkyne conjugation (stable) and streptavidin-biotin conjugation (stable).

Adequate tumor accumulation
Nanoparticles possess unique properties due to their small size termed as "enhanced permeability and retention effect" (EPR effect) ( Figure 2A) [29,30]. Harboring this phenomenon, nanoparticles extravasate through the leaky blood vessels (inter endothelial gaps as large as 500 nm) of the tumor vasculature and preferably accumulate at the tumor site. Due to poor lymphatic drainage, they retain at the tumor site. This phenomenon is also termed "passive targeting" (Figure 2A). Nanotherapeutics used for passive targeting are listed in Tables 1 and 11. However, targeting nanoparticles to the tumor tissue does not always remain successful due to variable vessel permeability [7].
In order to circumvent these barriers, researchers have taken advantage of conjugating tumor-targeting ligands to the nanoparticle surface resulting in "active targeting" ( Figure 2B) [7,41]. Binding of ligand to exposed cell surface results in "receptor-mediated endocytosis". However, receptor density plays a pivotal role in such targeting. Commonly used targeting agents are proteins (antibody and its fragments), ligands of the up-regulated receptors (peptides, carbohydrates and other small molecules) and nucleic acid aptamers [11]. Nanotherapeutics used for active targeting under clinical trial are mentioned in Tables 2 and 11. Peptides have been widely used as receptor targeting moieties for active targeting.   [11,43] Denileukin diftitox (Ontak) Interleukin 2 (IL-2) Diphtheria toxin fragment FDA approved Recombinant fusion protein of IL-2 attached to diphtheria toxin fragments [11,44] Ibritumomab tiuxetan (Zevalin) Mouse anti-CD20 antibody 90 Yttrium FDA approved Antibody-radioactive element conjugate [11,45] Tositumomab (Bexxar) Mouse anti-CD20 antibody 131 Iodine FDA approved Antibody-radioactive element conjugate [11,45]   Commonly used therapeutic agents are chemotherapeutic drugs, RNAi therapeutics, and imaging agents include MRI contrast agents, radionuclides, fluorescent probes, etc. Therapeutic agents can either be covalently conjugated or non-covalently encapsulated. Attaching imaging probes with nanoparticles containing therapeutic agents make it a theranostic platform (this image was drawn based on the information provided in Chou et al. 2011 [70], Figure 1).
Conjugation of ligands with the nanoparticles is mediated via covalent or non-covalent chemical conjugation. However, non-covalent conjugation often leads to weak bonding making it less efficacious. Covalent coupling is commonly achieved with the conjugations of the following groups (1) hydrazide-aldehyde, (2) carboxylic acid-primary amine, (3) thiol-thiol, (4) gold-thiol, (5) maleimide and thiol and (6) azide-alkyne. Different types of linkages formed due to various chemical conjugation reactions are described in Figure 3 [60,61].
Size, shape and surface modification also remain essential to achieve effective tumor targeting ( Figure 4). In order to specifically target the tumor, nanoparticles must first travel in circulation without being engulfed by macrophages [62]. Nanoparticles having a diameter of ∼5 nm undergo rapid renal clearance, whereas nanoparticles of 50-100 nm diameter mostly accumulate in the liver. Larger particles >2000 nm in diameter tend to accumulate in the spleen. It has been found that nanoparticles having a diameter of 100-200 nm can escape filtration by the liver and spleen ( Figure 5) [10]. Researchers observed that nanoparticles of size ranges 30-50 nm diameter showed maximum cellular internalization ability [63]. Studies have also shown that the size of nanoparticles is also dependent on tumor maturity. Reports suggest that with increasing particle size, the area of permeation within tumors become smaller [64]. Gold nanoparticles with 15-45 nm diameter have been found to accumulate in tumors of 0.5-1 cm 3 volume or above [65]. Recent findings suggest that the shape of the nanoparticles also determines the cellular uptake [66]. Several nanostructures like 2D polygonal shape, 3D polygonal shape, rod, snowflakes, flowers, thorns, hemispheres, cones, filaments, etc., have been designed to study their efficacies [62]. Recent studies have revealed that oblate shape is favoured in circulation [62]. Mitragotri and co-workers have modified a solid polystyrene microparticle into a red 6 Figure 5. Biodistribution of nanoparticles Nanoparticles having different (A) size, (B) shape, (C) surface charge and their biodistribution in different organs. This figure will guide us for designing organ-specific delivery of nanoparticles (this image was drawn based on the information provided in Blanco et al. 2015 [10], Figure 5). blood cell (RBC) shaped particle using layer-by-layer (LbL) self-assembly technique. The nanoparticles were synthesized using PLGA, which can be used as a carrier for drug and imaging agents [67]. Additionally, RBC-derived cell membrane and a hybrid membrane having membrane from RBC and cancer cell line were used for delivering chemotherapeutic drugs and such methodologies can be explored for designing personalized nanomedicine [68,69]. Surface charge of the nanoparticles also plays a significant role in nanoparticle internalization ( Figure 4). Current studies have demonstrated that nanoparticles having a size range of 50-100 nm carrying a very slight positive charge favour the penetration in large tumors [11].
A transient increase in blood pressure during systemic administration also causes increased tumor-specific nanoscale drug delivery. For in vivo delivery, near the wall margination is favored that interacts with the tumor vasculature bed. RBCs tend to travel in the middle of the blood flow, creating a "cell-free layer". Spherical nanoparticles follow the bloodstream whereas, rod-shaped nanoparticles undergo a lateral drift due to variable drag forces and torques. Nanorods show a 7-fold higher accumulation at the vessel lining than nanospheres. Again, discs marginate two times higher than rods. Particles are deposited at a higher rate at the site of the vessel bifurcation [62]. Additionally, the "multivalency" of a nanoparticle can be harnessed to enhance the binding affinity or avidity of ligand to receptor interaction [7]. It has been found that oblong-shaped nanoparticles are more helpful in forming more multivalent interactions compared to spherical nanoparticles ( Figure 6) [62]. The cellular internalization process also depends on another factor termed "membrane wrapping time" (Figure 7). Smaller nanoparticles tend to dissociate faster from receptors before being engulfed by the membrane to achieve receptor-mediated endocytosis. Again, extremely large nanoparticles limit the process of membrane wrapping. Chan and co-workers suggested that 40-50 nm nanoparticles remain the critical cut off point for receptor mediated endocytosis [71].

Controlled drug release
An ideal drug delivery platform should possess the ability to target and control the drug release, which facilitates sustained release of drugs [72]. Drug delivery often renders toxicity and side effects. These hurdles can be overcome by controlled drug release. This feature also leads to a high therapeutic index for the conjugated drug molecule. The binding of the drugs to the nanoparticles is achieved by adsorption, absorption, entrapment (the process of incorporation of a drug into a matrix) and covalent binding. The release of the drug molecules is decided on the basis of their solubility, desorption, diffusion through nanoparticles matrix, degradation of nanoparticle matrix and combination of such phenomena [73,74]. In many of such formulations, a phenomenon called "burst release" is often observed. A large bolus of drug is immediately released before it reaches to a stable profile. Researchers found that low molecular weight drugs are prone to exhibit burst release profile. Burst release may often lead to local or systemic toxicity, short half-life of drugs in vivo and shortened release profile [72]. Researchers have often modified the nanoparticles to get over this difficulty. Le and co-workers have used chitosan to modify PLGA nanoparticles carrying paclitaxel 8   and monitored the drug release profile. They found that modification of PLGA nanoparticles with chitosan led to reduced burst release of drug [75]. Again, designed nanoparticles that are prone to transcytosis (the vesicular transport of macromolecules from one side of a cell to the other) possess the potential to reach tumor cells and show efficient anticancer efficacy [76]. Nanotechnology has made advancements in mediating anticancer therapy and imaging at the tumor microenvironment. Several synthetic and natural nanoparticles have been used currently. These include polymeric conjugates and polymeric nanoparticles ((N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers, Poly(lactic co-glycolic acid) (PLGA) copolymers, etc.; liposomes and micelles; synthetic organic nanoparticles such as dendrimers; carbon-based nanostructures such as carbon nanotubes (CNTs) and polyhydroxylated/ PEGylated fullerenes; inorganic nanoparticles of gold, silver, and iron oxide; quantum dots (QDs); viral capsids and ferritin, etc [7]. Liposomes are spherical vesicles comprising of one or more lipid bilayers, especially phospholipids. Liposomes have been widely used as a drug delivery vehicle. These increase the drug's efficacy and therapeutic index by protecting the drug from the external environment. Dendrimers are radially symmetric branched polymeric nanoparticles. They possess the properties like poly valency, self-assembly, chemical stability, low toxicity and solubility. Dendrimers have been widely utilized to deliver anticancer drugs. Polymeric nanoparticles contain random or block co-polymers. These are colloidal particles having a size range of 1 to 1000 nm. Drug delivery has been widely performed using polymeric nanoparticles, either by encapsulation or chemical conjugation of drugs [18].
The delivery of chemotherapeutic drugs like cisplatin has been rigorously manipulated with nanotherapy to overcome their toxicity. Sengupta and co-workers have designed a novel cisplatin nanoparticle by harnessing PEG-functionalized poly-isobutylene-maleic acid (PEG-PIMA) co-polymer. Cisplatin is released in a pH-dependent manner and shows improved antitumor efficacy both in vitro and in vivo with limited nephrotoxicity [77]. They have also engineered another novel polymer glucosamine-functionalized PIMA to complex with platinum at a unique platinum to polymer ratio. Such nanoparticles also exhibit improved efficacy against breast and lung cancer with reduced systemic and nephrotoxicity [78]. Sengupta et al. have also designed cholesterol-tethered platinum II-based supramolecular nanoparticle with increased efficacy and reduced toxicity [79]. Prolonged use of cisplatin leads to nephrotoxicity. In order to overcome this limitation, other next-generation platinum-based drugs have been developed. Carboplatin (cis-diamino-(1,1-cyclobutandicarboxylate)platinum(II)) has been used recently, but it shows a cross-resistance with cisplatin. Oxaliplatin (cis-[(1R,2R)-1,2-cyclohexanediamine-N,N ]-oxalatoplatinum (II)) does not show such cross-resistance with cisplatin and is also highly soluble in water. Moreover, trans-1,2-diaminocyclohexane (DACH) ring of oxaliplatin adduct fills the major groove of DNA more efficiently than cisplatin. Scientists have derivatized the monomeric units of a PIMA copolymer with glucosamine, which chelates DACH platinum (II) and releases DACH-platinum in a sustained pH-dependent manner with reduced systemic toxicity and minimal kidney accumulation [80]. Kulkarni et al. also designed a computational algorithm to develop nanoscale supramolecular structures for cancer treatment [81].
Nanotechnologies have also been currently used for genetic treatments by nanoparticle-mediated delivery of RNAi therapeutics [10]. The primary challenge of RNAi-based therapeutics for its successful translation to clinics is the instability of RNA molecules, their rapid degradation in presence of nuclease and poor cellular uptake because of its highly anionic nature [82]. Genetic material, such as antisense oligonucleotides, mRNAs and siRNAs, and in the specific case of plasmid DNA have been used to achieve gene therapy via nanoparticles.
Peptides have been used for targeted delivery of diagnostics and chemotherapeutic agents for anticancer therapy. Peptides remain advantageous over other nanotherapeutics in terms of their self-assembling property, easy synthesis, structural manipulation to achieve protease stability, functionalization property, conjugation to the cell surface receptors and maximum therapeutic efficacy of cargo. Peptides show minimal toxicity, improved biodegradability, rapid renal clearance, and remain stable at physiological conditions. Anticancer agents like paclitaxel/docetaxel, doxorubicin, curcumin, fluorouracil have been successfully loaded on to self-assembled peptides and evaluated for their preclinical and clinical status [83]. Peptides have also been used as a potential molecular transporter based nanosystem to deliver RNAi therapeutics like siRNA [84][85][86]. Table 4 enlists cancer therapeutics that either have received FDA approval or are currently undergoing clinical

Nanoparticle in targeting oncogenic signaling pathways
Signal transduction pathways play a crucial role in cellular functions like survival, growth, differentiation and metabolism. In the case of cancer, such signaling pathways remain altered, leading to uncontrolled proliferation, immortality and tumorigenesis. Researchers have focused on identifying the drug targets to inhibit oncogenic signaling pathways [28]. Classically, humanized antibodies and small molecule inhibitors have been used as potential inhibitors to target such oncogenic pathways. But nanotherapy has helped target cancer cells selectively without causing toxicity to the healthy cells. The following section mentions key signaling pathways responsible for tumorigenesis and the approaches taken to target such pathways.
Targeting receptor tyrosine kinase (RTK) pathway for developing anticancer therapy [VEGFR], fibroblast growth factor receptors [FGFR], platelet-derived growth factor receptor [PDGFR] etc.). In cancer, RTKs are aberrantly activated and their mutations lead to various disorders. Therefore, RTKs and their ligands remain a potential drug target [28]. The inhibitors are mentioned in Figure 8. Kulkarni et al. found that multi-receptor tyrosine kinase inhibitor (XL184) loaded liposomes mediated nanoscale medicine improves intratumoral concentration, enhances antitumor efficacy and reduces toxicities [88].
Targeting the downstream signaling pathways also act as an alternative strategy. Mitogen-activated protein kinase (MAPK) and phosphatidylinositol-3-kinase PI3K-AKT-mTOR (mammalian target of rapamycin) pathways are two critical downstream pathways [28].
Targeting mitogen-activated protein kinase (MAPK) signaling for developing anticancer therapy MAPK pathway comprises a series of proteins like rat sarcoma virus (RAS), rapidly accelerated fibrosarcoma (RAF), mitogen-activated protein kinase kinase (MEK) and extracellular signal-regulated kinase 1/2 (ERK 1/2). This pathway is up-regulated in most cancer types and responsible for abnormal proliferation leading to tumorigenesis. RAS, RAF and MEK gained the most attention as therapeutic targets [28]. The progress in the therapeutic strategy targeting MAPK pathway using small molecule inhibitors is mentioned in Figure 8.

Targeting phosphoinositide 3-kinase (PI3K) pathway for developing anticancer therapy
Phosphoinositide 3-kinase (PI3K) pathway is responsible for cellular processes like proliferation, growth, survival and apoptosis. This pathway is mutated in 30% of all human tumors. Activated RTKs set off several downstream signaling cascades, especially protein kinase B (Akt) and mammalian target of rapamycin (mTOR), responsible for various cellular functions [28]. The small molecule inhibitors targeting this oncogenic signaling pathway are mentioned in Figure 8.
Utilizing the nanotechnology, Harfouche et al. found that encapsulating LY294002, a PI3 kinase inhibitor into biodegradable PLGA nanoparticle causes successful inhibition of Akt phosphorylation resulting in the inhibition of proliferation [90]. Sengupta and co-workers have rationally conjugated PI3K inhibitors (PI103 and PI828) using a cholesterol-based derivative, enabling supramolecular nano assembly with L-α-phosphatidylcholine and DSPE-PEG [1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polythylene glycol)] to achieve increased antitumor efficacy [91]. They have also suggested that a rational combination of cis-platinum nanoparticles and a PI3K-targeted therapeutic remains a potential therapy for breast cancer [92]. Free nanoparticles or nanoparticles carrying different therapeutic agents like oligonucleotides, cytotoxic drugs, antibodies, etc., have been used to target different oncogenic pathways (Table 5).
Another approach to target oncogenic signaling pathways is mediated by siRNA delivery, a component of gene therapy. Peptides have also been used as a molecular transporter-based nano-system to deliver siRNA targeting signaling pathways [86,105,106,107]. This is discussed in the later section of this review.

Nanoparticle in tumor angiogenesis
Angiogenesis is a phenomenon of the formation of the new blood capillaries out of pre-existing blood vessels. This phenomenon is essential for wound healing [108][109][110][111]. Cancer metastasis is also positively correlated with angiogenesis [112]. Therefore, blocking angiogenesis is a practical approach to inhibit cancer progression. Commonly available anti-angiogenic therapy harbors any of these two strategies: (1) damaging the existing blood vessels or (2) preventing the formation of new blood capillaries [113]. Anti-angiogenic therapy is different from commonly used tumor-targeted chemotherapy. It selectively targets the tumor-associated vasculature instead of the tumor cells, shows increased bioavailability for systemically administered drugs compared with tumor-targeted therapy and requires a low administration dose, leading to lesser systemic toxicity [113,114].
In spite of these advantages, chemotherapy-mediated anti-angiogenic therapy suffers from certain limitations. The tumor vasculature remains unreachable to most of the anti-angiogenic inhibitors. Nanotechnology has become an advanced and effective method to address this problem. Nanoparticle-mediated delivery of therapeutics has been achieved following both passive and active targeting [113]. Researchers have shown that conjugating cytotoxic drugs or angiogenic inhibitors with nanoparticles prefer to home at the tumor site following the EPR effect Kupffer cells Inhibition of NF-κB activation by D-NPs in kupffer cells (KC) D-NPs inhibit the nuclear translocation of p65, a common subunit of NF-kB. Improve survival and reduction in liver damage [93] Folate-linked lipid-based nanoparticles NF-kB decoy RAW264.7 The NF-κB decoy shows an inhibitory effect in cytoplasm (inhibition of NF-kB translocation into nucleus of LPS-activated macrophages) Inhibit particular matter (size ≤2.5μm) (PM2.5) induced neuroinflammation [94] Fisetin nanoparticles (FN 14  [115,116]. Active targeting has also been effective in targeting angiogenesis. Anti-angiogenic therapy harbors targeting VEGFR, α v β 3 integrins and other angiogenic factors. Synthetic peptides having the recognition site for integrins (cyclic Arginine-Glycine-Aspartic acid [cRGD]) have been widely used for targeted nanotherapy [113]. A plethora of nano-vectors have been reported by researchers for nanotechnology-based anti-angiogenic therapy, including polymeric nanoparticles, liposomes and micelles, dendrimers, carbon nanostructures, inorganic nanoparticles (e.g., gold, silver and iron oxide), etc. Recent nanotechnology-based anti-angiogenic therapies also use a gene silencing-based approach using therapeutic genes or siRNA [113].
A few reports on nanotechnology-based anti-angiogenic therapy are provided in Table 6.

Nanoparticle-mediated gene therapy for cancer
Gene therapy is the modulation of gene expression towards treating a disease by cellular delivery of therapeutic nucleic acid. It holds unique promise in alteration of specific tumor genes functioning via gene addition, gene correction or gene knockdown [16]. Several approaches of cancer gene therapy include (1) suicide gene therapy: introduction of an enzyme expressing transgene into the cell that converts inactive prodrug into cytotoxic metabolite for host cells, (2) gene silencing: suppression of gene expression by RNAi techniques like siRNA, shRNA, antisense oligonucleotide, miRNA, etc., and (3) DNA vaccine: introduction of specific antigen encoding plasmid DNA into the cell to induce immune response. Gene therapy shows the potential to deal undruggable targets to treat cancer as compared with conventional treatment by targeting cancer associated genes. Gene therapy can provide a solution to low bioavailability, reducing immune system based recognition and delivery of the gene regulators [131]. The fundamental challenge in the engineering of gene therapy is the development of clinically safe and effective delivery vectors. In clinics, both viral and non-viral mode of delivery is being used for systemic gene delivery [132]. The viral delivery systems are associated with various safety concerns as well as limited payload capacity and difficulty in large-scale production [16]. These factors led to the development of interest toward non-viral synthetic vectors for gene therapy. The non-viral vectors are advantageous in providing higher safety profile, low cost, large scale manufacturing potential, stability and higher payload [132]. Nanoparticle and nanoscale gene delivery vectors have emerged as efficient candidates for intracellular or systemic gene delivery.

Lipid-based nanoparticles
Lipid-based nanoparticles are the most widely used non-viral gene delivery vehicle [132,133]. Cationic liposomes are amphiphilic molecules which are made up of cationic polar head group, a hydrophobic domain and a linker connecting the polar head group with the non-polar tail. This cationic liposomes are routinely used for gene delivery [134]. Incorporation of longer lipidic chain (having around 18 methylene group, which can span the whole membrane) having unsaturation and small polar head facilitates the formation of conical shaped lipid in anionic membrane environment, which promotes the hexagonal phase transition of the lipid bilayer from the lamellar phase [135]. Hexagonal phase of the lipid bilayer is more fusogenic than lamellar phase. This transition of lamellar phase to hexagonal phase of lipid bilayer leads to cellular internalization and endosomal release of the internalized lipid-based nanoparticle [135]. Lipid nanoparticle containing ionizable lipids are designed in a way that it gets protonated at endosomal pH range (4.5-6.5). The protonated cationic lipid interacts with the anionic lipid of the endosomal membrane, leading to the transition of the lamellar phase to the hexagonal phase of lipid bilayer. Several liposomal nanoformulations have been in clinical development like DOTAP-cholesterol, GAP-DMORIE-DPyPE, etc. The FDA approval of Alnylam's patisiran in 2018, is the first ever drug to successfully harness RNA interference to silence disease associated gene expression was a key milestone. Further, FDA approved lipid based nanoparticles like givosiran, lumasiran established a benchmark for lipid nanoparticle based drugs. Hou et al. have discussed in detail about the use of lipid based nanoparticles for gene delivery, their drawback and engineering principles for engineering next-generation improved lipid based nanoparticles [136].

Polymer-based nanoparticles
The chemical diversity and functionalization potential of cationic polymer based nanoparticle makes it an attractive class of non-viral gene delivery vehicle. Polymers are considered as inert but certain modification by biologically active agents and counterions like spermidine or cell penetrating peptides improves the surface functionalization, nucleic acid loading and particle transfection [16] PLGA nanoparticle is capable of delivering nucleic acid with minimal toxicity but exhibit low transfection efficiency [16]. PLGA nanoparticles are surface functionalization with cell targeting or cell penetrating peptides to improve nucleic acid loading and cell penetrability. The condensation of negative phosphate bonds of nucleic acid with the cationic polymers into polyplexes protects nucleic acid from degradation during circulation [16].
Polyethylenimine (PEI) and its variants are among the most studied polymeric materials for gene delivery. The presence of nitrogen at every third position of the polymer increases the charge density and reduces the pH [132,137]. Various polymer-based nanoparticles such as dendrimers, polyion complex micelles (PICs), cyclodextrin, etc., have also been explored greatly in gene therapy. The polymeric nanoparticle system owing to their facile synthesis and flexible properties proves to be a new promising material for developing non-viral gene delivery system. The cationic polymer can be conjugated with the negatively charged genetic material via electrostatic attraction at physiological pH and facilitating gene delivery. Ekladious et al. have discussed in detail about the rational designing, physicochemical characteristics and advancements in different classes of polymer based delivery vehicle and their application in different fields including gene therapy [138].

Peptide-based nanoscale material
Cell penetrating peptides (CPP) are a class of peptides facilitating the cellular internalization of nucleic acid based therapeutics either by covalent or non-covalent conjugation. The cellular uptake of the CPP peptide depends upon their sequence, structure, concentration and cell lines used for study [139]. The cellular internalization of cell penetrating peptides follow either by one or by the combination of the following mechanisms: (1) creating a transient pore in cell membrane, (2) endocytic uptake and (3) receptor mediated uptake. Depending upon the sequence, the CPP can be of cationic CPP or amphiphilic CPP.
The cationic CPPs are arginine or lysine rich short peptides. Arginine rich sequences are found to show enhanced cellular uptake. The arginine can form a more efficient bidentate hydrogen bond than the monodentate hydrogen bond formed by lysine with the anionic moieties like phosphate (PO 4 3− ), carbonate (CO 3 2− ) and sulfate (SO 4 2− ) on the cell surface [140]. These interactions facilitate the cellular internalization of the arginine or lysine residue enriched cationic peptides.
Amphiphilic peptides show enhanced cellular uptake by the formation of lipid rafts in the cell membrane. The cationic part of the CPP interacts with the negatively charged therapeutic nucleic acid and the hydrophobic part facilitates the cellular internalization of the peptide by interacting with the lipid bilayer [141,142].
N-Methylpurine DNA Glycosylase or MPG (GALFLGFLGAAGSTMGAWSQPKKKRKV) peptide is a great example of amphiphilic CPP. The hydrophobic stretch of this amphiphilic CPP (underlined) adopts a transient β-sheet structure creating a temporary channel in the cell membrane allowing the peptide-nucleic acid complex to internalize [143,144]. Amphiphilic α-helical peptide like penetratin (RQIKIWFQNRRMKWKK) generally remains unstructured in an aqueous solution but tends to adopt an α-helical conformation while interacting with cell membrane [84]. Majority of CPP internalizes via endosomal pathway and are accumulated inside endosome. Two strategies employed for endosomal escapes are use of (1) conformation changing fusogenic peptides and (2) proton buffering peptides [84]. The conformation changing fusogenic peptides in endosomal pH (4.5-6.5) undergoes a conformational change and ruptures the endosomal membrane by forming an amphiphilic helix [84]. These peptides are rich in histidine and glutamic acid and are sensitive to pH change. pH sensitivity arises due to imidazole group of histidine side chain having pKa value of ∼ 6.0 and glutamic acid side chain with pKa ∼ 4.3 allowing easy protonation and de-protonation of these amino acids in endosomal environment.
Proton buffering peptides escape the endosomal entrapment by accumulation of proton absorbing peptides inside endosome and disrupting the endosomal membrane by proton sponge effect. Imidazole ring of histidine having a pKa value of nearly 6.0 shows high buffering effect by acting as a weak base inside endosome. This causes influx of protons into the endosome and osmotic swelling causing endosomal rupture. Peptides provide advantage as a delivery vehicle due to their biocompatibility, biodegradability and sheer limitless combinations and modifications of amino acid residues inducing the assembly of modular, multiplexed systems [145]. Tarvirdipour et al. have discussed in detail the designing principles and attractive features of peptide based nanoscale materials for gene therapy [145].

Inorganic nanoparticles
This includes carbon nanotubes, magnetic nanoparticles, calcium phosphate nanoparticles, gold nanoparticles and quantum dots that are commonly used for gene delivery vehicles. The inorganic nanoparticles are resistant to microbial attacks and provide good storage stability [146]. The functionalized single-walled nanotubes are reported to enter premyelocytic leukemia and T-cell very easily, this ability is exploited to deliver nucleic acid into mammalian cells [147]. Water soluble allotrope of carbon (C60) fullerenes modified to aminofullerenes has positive charge on them. This aminofullerenes are reported to have high transfection efficacy of DNA into mammalian cells [148]. Inorganic nanoparticles are reported to have great gene delivery efficacy on surface modification. Chen et al. have discussed in great detail about the use of inorganic nanoparticle as a drug codelivery nanosystem [149]. Table 7 lists out some of the important nanoparticle based gene therapeutic approaches. Advancement in clinical studies of engineered siRNA-loaded nanoparticles has been discussed in Table 8 [150,151].

Nanoparticle-mediated combination therapy against cancer
Combination therapy, a treatment modality involving combination of two or more therapeutic agents, has now become the cornerstone of cancer therapy since single drug based monotherapy failed to provide a considerable therapeutic response. Enhanced therapeutic potential, reduced toxicity and prevention of drug resistance mediated by combination therapy have hugely increased the therapeutic response of cancer patients. Combination therapy can be designed in two different ways. First, targeting of different molecular pathways by multiple drugs can lead to delayed cellular adaptation and oncogenic mutations. Second, targeting the same pathway could be helpful in the development of synergistic interaction of multiple drugs with higher efficacy and target selectivity. Synergistic interaction is best described by the Chou-Talalay method where combination index (CI) is less than 1 [167,168]. Boshuizen et al. suggested that a synergistic drug interaction in a combination therapy can be rationally designed by invoking several principles: (1) multiple targeting of a signaling pathway, (2) maximal driver pathway inhibition, (3) targeting agents responsible for signal reactivation, (4) enhanced synthetic lethality, (5) targeting heterogeneous and drug resistant cell populations in a tumor, (6) targeting immune cell function and tumor microenvironment modulation and (7) neoadjuvant therapies [169]. Combination therapy also employs repurposing of drugs that were initially used for diseases other than cancer. This approach saves the high cost and time required for granting FDA approval as the repurposed drug is generally an FDA approved drug, thus cutting the cost of the treatment [170]. The synergistic agents involved in modern combination therapies should ideally have a different pharmacological mechanism of action, exhibit no cross-resistance or overlapping toxicities and target tumor heterogeneity [171]. By integrating the knowledge and progress made in the field of mechanisms responsible for tumorigenesis, tumor microenvironment, therapy response and cancer heterogeneity, an effective treatment can be designed [169].
Nanoparticles have emerged itself as a potent tool to be used in combination therapy as they provide longer circulation time of biologically active drug, reduced toxicity, improved drug solubility, controlled release and has specific target potential [172]. Gurunathan and co-workers have elaborately described different types of nanoparticles mediated combination therapies [173]. The rationale for designing nanoparticle-mediated combination therapy for cancer treatment is provided in Figure 9. In Table 9, we have enlisted examples of nanoparticle-mediated combination therapy for treating different types of cancer and their advantages over monotherapy.
In recent years, there has been an increase in the number of reports of multifunctional nanoparticle mediated combination therapy aided with peptides. Some of the advantages of peptide-mediated combination nanotherapeutics are tumor targeted delivery, presentation of tumor antigens for elicited immune response, sensitization of drug resistant cells and reduced side effects [187]. Mehrotra et al. and Mallick et al. have elaborately enlisted different types of peptide-based combination nanotherapeutics [187,188]. We have enlisted some of them in Table 10.
However, the current combination therapy treatment in cancer, especially in the case of metastatic breast cancer, is still loaded with flaws having moderate efficacy but additive toxicity [18]. Drugs, when administered separately without any modification, tend to not only cause an additive anticancer effect but also result in augmented adverse effects of each drug as well. For achieving synergistic interactions, a definite ratio of the two free drugs needs to be maintained. This ratio is generally determined in in vitro studies, and it is crucial that this ratio is to be maintained at tumor site. The problem arises because in in vivo model it becomes virtually impossible to deliver the determined ratio of free drugs in the tumor site. This is mainly due to the different pharmacokinetic properties, elimination and metabolism rates of individual drugs. Delivery of non-fixed ratio of drugs can give rise to antagonistic interactions and drug resistance in cancer cells [257]. For example, irinotecan and cisplatin (both cytotoxic drugs) show synergistic interaction at 4:1 ratio but strong antagonistic interaction at 1:1 ratio and in the case of many anticancer drugs, when the effective dose is below its optimum dose, it may give rise to drug resistant cells in tumor [257]. Another complication arises when the free drugs need to be administered by different routes and at different schedules. HER2 targeted combination therapy with Trastuzumab (TRZ, a monoclonal antibody binding to HER2 receptor) and lapatinib (a tyrosine kinase inhibitor blocking HER2 and EGFR pathways) have two different routes of administration. Lapatinib is administered daily as an oral formulation, while TRZ is given weekly as an intravenous drug. This difference in schedules and ways of administration of these two drugs makes the management of pharmacokinetic and pharmacodynamic profiles more challenging and virtually impossible to achieve uniform temporal and spatial co-delivery [18]. Most standard chemotherapy guidelines prefer sequential delivery of free drugs over concurrent delivery due to toxicity issues. This also prevents the right temporal delivery of drugs with dissimilar pharmacokinetic properties [258]. To address these issues, nanoparticle-mediated co-delivery of those drugs may ensure the desired spatio-temporal delivery and controlled release of drugs while maintaining the synergistic fixed ratio ( Figure 10) [258]. Nanocarrier mediated co-encapsulated drugs having physically different properties show similar pharmacokinetic profiles, extended drug half-life, solubility, tumor accumulation and synergistic drug interactions when compared with free drugs administered sequentially or concurrently [18,257]. Cytrabine (an antimetabolite chemotherapeutic drug) and danorubicin (a cytotoxic chemotherapeutic drug) has distinct pharmacokinetic properties as free drugs but when co-encapsulated in liposomal nanocarrier as Vyxeos ® exhibit similar pharmacokinetic profile [257]. Nanocarrier mediated co-delivery of drugs enables targeting multiple signaling pathways, overcoming drug resistance and immunosuppression by cancer [257].
Various delivery systems widely used for co-delivery of two drugs include liposomes, dendrimers, polymeric nanoparticles and water-soluble polymer conjugates. Lee et al. and Gurunathan et al. have elaborately reviewed nanoparticle-mediated co-delivery of two or more drugs for cancer therapy as described in Table 11 [18,173].
In some instances, co-delivery of drugs in a single nanoparticle suffers from drug leakage and poor loading efficacy [259]. To overcome this problem, researchers have conjugated two anticancer drugs via suitable covalent linkages and then subsequently encapsulated the conjugate in a nanoparticle. For example, Aryal et al. have used ester linkage for conjugating paclitaxel and gemcitabine and loaded the drug conjugate into a PLGA nanoparticle. Hydrolysis of this ester linkage at mildly acidic pH (pH 6) of endosomal environment resulted in two separate functional drug fragments [260]. Matlapudi et al. have conjugated Imatinib mesylate (abbreviated as IM, a tyrosine kinase inhibitor) and 5-fluorouracil (abbreviated as FU, an antimetabolite) by hydrolysable succinyl linker (abbreviated as Su) which forms amide linkage with each drug. This drug conjugate IM-Su-FU was incorporated in a human serum albumin (HSA) nanoparticle. This HSA encapsulated drug conjugate (IM-Su-FU) nanoparticle exhibited higher anticancer efficacy in in vivo lung cancer model compared to free drugs and only IM-Su-FU conjugate. Pharmacokinetic analysis of this nanoparticle exhibited improved elimination rate, half-life and mean residence time (MRT) than free drug and only IM-Su-FU conjugate [261].     [190,191] Acetylated rapeseed protein isolate derived peptides + DOX From hydrolyzed ARPI peptides 3 bioactive peptides were screened. Sequences are AGS, PAS and YT. DOX-cytotoxic

Challenges and future prospect of nanoparticle-mediated combination therapy
Nanoparticle-mediated combination therapy has shown great potential in treating metastatic and drug resistant cancer [262]. Combination therapies involving cytotoxic drugs, signal transduction inhibitors, immunotherapeutic drugs, epigenetic agents and priming with apoptotic drugs have shown promising possibilities for cancer therapeutics [258]. The success of nanoparticle mediated combination owes to its "3R" delivery principle, i.e., right place, right dose and right time. To gain the benefits of nanocarrier mediated combination therapy, different databases and drug development platforms are developed. EMBASE ® and Ovid MEDLINE ® are databases used for the co-delivery of drugs. The drug combination development platform CombiPlex screens dual drugs and makes nanoscale formulation for nanocarrier mediated co-delivery of drugs. CombiPlex platform first determines the synergistic ratio of free drugs and then chooses a suitable nanocarrier to coordinate the pharmacokinetics of the free drugs and ensures the drugs reach the tumor site in the desired ratiometric manner. CPX-351, developed from CombiPlex platform is an FDA approved drug used for treating AML in adults. CPX-351 comprises cytarabine and daunorubicin in a synergistic ratio encapsulated in a liposomal carrier [257]. Although nanoparticle-mediated combination therapy has immense potential, but the regime of nanoparticle-based combination therapy is far from being able to cure metastatic cancers. 0.7% is the median efficiency of delivery of the injected nanoparticle to the desired tumor site [24]. Additionally, the five-year survival rates of most malignant cancer are still quite low and most combination approaches still depend on cytotoxic approaches instead of molecularly targeted anticancer agents or nanoparticle mediated combination approach [263]. Saptura et al. has shown antagonistic drugs interactions used in combination can suppress the clonal expression of singly-resistant cells in in silico model [264]. So antagonistic drug interactions should also be investigated and not completely discarded. Although around five thousand clinical trials are ongoing worldwide for the development of new combination therapy, Palmer and Sorger, 2017 claims that most of the combination therapy used follows the 24      [173,256] independent action model and provide therapeutic benefit due to patient-to-patient variability rather than additive or synergistic drug interactions [265,266].
One of the key challenges in developing nanoparticle-based targeted therapy is the issue of toxicity of treatment combinations. Seemingly rationally developed and assumed to be safe combination therapy in preclinical model may fail in clinical trials due to toxicity, especially in the case of synergistic drugs which can lead to synergistic toxicity as well due to similar mechanism of action or due to auto immune response of healthy tissue [169]. Mathematical modeling has been helpful in predicting toxicity in pre-clinical trials and can be used as an efficient tool for predicting the toxicity of drug combinations in a patient-specific manner [169]. Toxicity of the drug nanocarrier also needs to be considered. Another hurdle in the success of combinatorial nanotherapeutics to be translated into clinics is the lack of patients who are interested in participating in clinical trials. Approximately 40% of cancer trials fail due to the scarcity of patients [169]. Cancer is a highly heterogeneous disease and cell type population in a particular type of malignant tumor varies from patient to patient. For clinical trials to have higher success rates, efficient biomarkers need to be identified first. Additionally, clinical trials should be performed on patients with a similar level of biomarker expression. But due to the lack of patients enrolled in a clinical trial, such arrangements are not possible. Due to the insufficiency of suitable patients for performing clinical trials, precise preclinical models need to be developed. Researchers need to look beyond immune-competent mice model and use patient-derived xenograft in vivo models, humanized mouse models and patient-derived organoids for a greater chance of success in clinical trials and such models can also be used as an efficient tool for predicting the toxicity of drug combinations in a patient specific manner [169].

Nanomedicine and combination nanomedicine for cancer immunotherapy
Immunotherapy is a monumental breakthrough recently included in the existing therapeutic armamentarium against cancer. It utilizes body's own immune system to fight cancer. William Coley, known as the father of immunotherapy, first attempted to treat cancer utilizing the immune system. However, this field got attention when James P. Allison and Tasuku Honjo got the Nobel prize for cancer immunotherapy in the year 2018. Judy Perkins having stage IV metastatic breast cancer, was the first lady to be cured successfully by immunotherapy [267]. Current cancer immunotherapy comprises cytokine therapy, antibody-based therapy and adoptive cell therapy. However, complex tumor microenvironment limits the efficacy of immunotherapy. In the tumor microenvironment, tumor cells can polarize tumor associated macrophages (TAM) toward pro-tumorigenic M2 macrophages while decreasing antitumorigenic M1 macrophages to facilitate tumor progression. Moreover, cancer cells help to activate immune checkpoints leaving the T cells in a state of anergy (lack of responsiveness to an antigen). Cancer immunotherapy inhibits immune checkpoints to overcome the T-cell anergy, thereby its activation against cancer. Ipilimumab inhibits the cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) checkpoint in T cells leading to its activation. Similarly, programmed cell death protein 1 (PD-1) in T cells is inhibited by FDA-approved anti PD-1 antibody pembrolizumab and nivolumab. Cancer cells overexpress the immune checkpoint protein programmed death-ligand 1 (PD-L1). The PD-L1 inhibiting antibodies atezolizumab, avelumab, durvalumab are also approved by FDA [268]. These antibodies block the inhibitory signal induced by interaction of PD-1 of T cell to PD-L1 of the cancer cell. Multiple colony stimulating factor receptor 1 (CSF-1R) inhibitors are currently in clinical trials for their ability to polarize tumor associated macrophages to anti-tumorigenic M1 phenotype [13].
Peptide-based drugs/vaccines can hugely advance cancer immunotherapy. Peptides can be designed in a more rationalized manner to target desired molecule of tumor or the tumor microenvironment. Yin et al. have designed a peptide (IQIREYKRCGQDEERVRRECKERGERQNCHYVIHKEGNCYVCGIICL) mimicking the native structure of the PD-1 molecule, which inhibited the interaction between PD-L1 of cancer cells and PD-1 of immune cells. This peptide was developed as a potent drug for cancer immunotherapy [269]. Hazama et al. also developed a peptide-based macrocyclic peptide (c[Ac-D YRYSAVYSIHPSWC]G) inhibiting the interaction between cluster of differentiation 47 (CD47) of cancer cell and signal regulatory protein α (SIRPα) of macrophages and showed its in vivo efficacy [270]. Peptide-based cancer vaccines can specifically stimulate cancer specific T cell response [271]. Peptide-based (9 amino acid residues) vaccine GP2 (IISAVVGIL) is currently in phase 2 clinical trial against breast cancer [272]. Several other examples of peptide vaccines combined with other drugs are highlighted in Table 10.
Researchers have reported a number of combinatorial approaches where immunotherapeutic drugs are combined with the conventional cancer therapeutics such as chemotherapy, RNAi therapy, photothermal, photodynamic and radiotherapy as described in Tables 12-14 and Figure 11.

Chemotherapy with immunotherapy
Combination chemotherapy with cancer immunotherapy is a wide area of interest. In phase III clinical trial, albumin nanoparticle bound chemotherapeutic drug paclitaxel (Abraxane ® ) combined with atezolizumab (Tecentriq ® ; FDA approved antibody of PD-L1) has recently demonstrated its efficacy in patients with advanced triple-negative breast cancer (TNBC) [22,298]. However, some patients showed grade 3 (moderate to severe) or grade 4 (life threatening symptoms) immune related adverse events [298]. Hence, combination of nanoparticle-mediated chemo-immunotherapy may facilitate targeted and localized delivery to increase the therapeutic index, reduce off-target toxicity and incidences of immune related adverse events. Tables 12 shows some examples of preclinical studies on combination of nanoparticle mediated chemotherapy and immunotherapy.

RNAi therapy with immunotherapy
RNA interference (RNAi) therapy with siRNA, microRNA (miRNA) and short hairpin RNA (shRNA) is utilized to silence genes of specific signaling molecules, cytokines and chemokines. However, this therapy possesses certain limitations like rapid degradation of RNA-based drugs in circulation due to presence of nucleases, renal clearance, poor cellular uptake because of anionic nature, etc [299]. Therefore, nanoparticles have been used to circumvent such barriers. Gene therapy has been utilized in targeting immune checkpoints like PD-1-PD-L1 pathway [300]. Also, these can be used with other cancer treatment modalities to induce immune stimulation against cancer. Few examples are given in Table 13 where combination nanomedicine having immunotherapeutic drugs showed enhanced efficacy.

Photothermal, photodynamic and radiotherapy with immunotherapy
Photothermal, photodynamic and radiotherapies are also used in combination with immunotherapy. In photothermal therapy, cancer cells can be destroyed and eliminated from the tumor tissues at 40-44 • C temperature due to DNA damage, protein denaturation and disruption of cellular membrane [20]. However, it requires high temperature for complete cell death, thus large tumors are prone to relapse. Thus combining photothermal therapy with immune-stimulating agents and nanoparticles can synergistically induce anti-tumor efficacy for the treatment of large established tumors and distant metastases.
Photodynamic therapy comprising combination of light with photosensitizers leads to the generation of reactive oxygen species (ROS) and subsequently damage of subcellular organelles [301]. However, as a monotherapy, it does not efficiently regress the tumor in an immunosuppressive tumor microenvironment [20]. It induces immunosuppression by the release of immunosuppressive cytokines. It provokes damage to the normal cells by releasing the Significant tumor growth and metastasis inhibition [282] self-antigens [302]. Thus, to balance the immunosuppressive effects of mono-photodynamic therapy, immunotherapy needs to be combined with it.
In radiotherapy, high energy ionizing radiation such as X-rays are utilized to cause free radical generation mediated DNA and cellular damage leading to cellular death [303]. However, radiation therapy helps to develop anticancer therapy, its solo use promotes immunosuppressive environment around tumor by recruiting immunosuppressive T reg cells [304]. It causes increased production of immune-inhibitory molecules such as PD-L1 and transforming growth factor-β (TGFβ) [305]. Thus, to overcome these problems of radio-monotherapy, combination of radiotherapy with immunotherapy is implicated. Some examples are given in Table 14.

Next-generation combination nanomedicine for immunotherapy
Immune checkpoint inhibitors were initially used in combination with other conventional chemotherapeutic drugs or radiotherapy (Tables 12-14) to potentiate the tumor regression. At present, nanomedicines for immunotherapy combines mechanistically inspired drug which can demonstrate a synergistic effect. Sengupta and co-workers have designed a supramolecular bifunctional nanomedicine comprising amphiphiles which inhibits interaction of CD47 of cancer cell and SIRPα of macrophages and simultaneously the interaction of macrophage colony stimulating factor (MCSF) and colony stimulating factor 1 receptor (CSF-1R). This nanomedicine increases the M2 to M1 repolarization within the tumor microenvironment and improves the anticancer and antimetastatic efficacy in melanoma and breast cancer model [306]. Mitragotri and co-workers designed a class of phagocytosis-resistant discoidal particles containing interferon-γ (IFN-γ). These particles showed efficient adherence to macrophages and directed their polarization toward anti-tumor M1 phenotype in murine breast cancer model [307]. Other such combinations are described in the next section.

Combination immunotherapy with signaling pathways inhibitors
Next-generation cancer immunotherapy needs to be patient-specific to enhance its antitumor efficacy. Immunotherapeutic drugs can be combined with signaling pathway inhibitors for better synergistic outcomes and to amplify the efficacy of personalized medicine [308]. MEK inhibitors are used in combination with PD-L1 antibodies as MEK inhibition causes up-regulation of PD-L1 in cancer cells. In cancer, the dysregulated PI3K-AKT pathway also regulates the PD-L1 expression [309]. Kulkarni et al. designed a nanomedicine combining PD1-PDL1 immune checkpoint inhibitor with kinase (MEK and PI3K) inhibitors for enhanced anti-tumor efficacy [81]. It has also been reported that second generation anti-histaminic drugs like cetirizine, etc., in combination with immunotherapy, have improved the anticancer efficacy [310].

Combination immunotherapy with GPCR inhibitor
Combination of G-protein-coupled receptor (GPCR) inhibitors and immune checkpoint inhibitors can amplify antitumor efficacy. CXCR4, a GPCR, is overexpressed in cancer cells which mediates cell proliferation, tumor growth, metastasis and tumor relapse [311]. CXCL12, the chemokine binding to the CXCR4, is secreted from fibroblast cells in the tumor microenvironment. CXCL12/CXCR4 axis is another molecular target for cancer treatment as this axis leads to an immunosuppressive tumor microenvironment. Thus, combining plerixafore (AMD3100; CXCR4 inhibitor) with immune checkpoint inhibitor anti-PDL1 antibody showed decreased tumor volume [312]. Eliminates NIR laser exposed tumors but causes strong anticancer immunity to inhibit distant tumors also [294] TBC-Hf (derived fromn tetra(pbenzoato)chlorin and Hf) enabled photodynamic therapy

Combination immunotherapy with epigenetic drugs
Epigenetic drugs can potentiate combination immunotherapy [313]. DNA hypomethylating agent 5-aza-2 -deoxycytidine (5-AZA-CdR) (a DNA methyltransferase inhibitor, DNMTi) combined with an anti-CTLA-4 monoclonal antibody showed enhanced antitumor efficacy in murine model of breast cancer [314]. In Phase Ib clinical trial, the epigenetic drug guadecitabine was combined with ipilimumab (anti-CTLA-4 antibody) for patients having stage III/IV melanoma. The DNA hypomethylating drugs cause upregulation of genes CD274, PDCD1LG2, and CTLA-4 in the patients where resistance has developed. Currently, a PEG-based nanoparticle system was utilized to deliver plasmid-encoding shPD-L1 (this plasmid down-regulated expresion of PD-L1 protein of cancer cells) in combination with Zebularine (a DNMTi, causing overexpression of major histocompatibility complex I [MHC-I] expression). This combination can effectively initiate anticancer immunity and prevent tumor relapse by a strong anticancer memory [315].

Limitations and future prospect of nanoparticle-mediated combination immunotherapy
Currently, immunotherapy is the latest approach that has emerged in finding a cure for cancer. However, only immunotherapy is insufficient to eradicate tumors due to : (1) lack of specificity and systemic toxicity, (2) low patient response rate, (3) variable immune contexture of patients, etc., (4) impaired immune function and antibody activity due to the acidic tumor microenvironment (Warburg effect), (5) divergent immune pattern of different organs, (6) development of resistance against immunotherapy, (7) autoimmune adverse effect generation by immune checkpoint blockade, (8) increased immune suppression with aging and (9) huge cost [316][317][318].
To advance the area of cancer immunotherapy in near future, toxicity issues associated with immunotherapy need to be addressed. Since cancer is a highly heterogeneous disease, nanocarrier encapsulated precision combinatorial immunotherapy will hold a great prospect in future cancer therapy. Tumor heterogeneity can be explored by the determination of biomarkers via genomic analysis of circulating cancer cells. Next-generation sequencing can be deployed to explore immunogenicity of mutated genes. These data along with resources like cBioPortal, Project Genie can be utilized in designing patient-specific immunotherapy in an effective and safer manner. Immunotherapy can be further boosted by utilizing combinatorial approaches. This would require identification of optimal dosing and delivering the same dose to the tumor site. This can be achieved by ratiometric dosing as described earlier. Mono anti-PD-1 therapy often causes compensatory activation of other checkpoints (e.g., CTLA-4), leading to immunosuppression [319]. Thus, simultaneous blocking of inter-related checkpoints is necessary to design effective combinatorial immunotherapy. The gut microbiome or blood supply to the tumor site can also alter responses to cancer immunotherapy by indirectly modifying tumor microenvironment [320]. Hence modulating these factors can help combating resistance to immunotherapy. Furthermore, antagonizing low pH of tumor microenvironment by acidity modulating drugs (proton-pump inhibitors) might act as a possible choice to overcome tumor resistance, potentiating the existing immunotherapy [318]. Thus, optimizing the current immunotherapy with personalized combinatorial nanomedicines can advance the development of next-generation cancer nano-immunotherapy.

Conclusion and future direction
The challenges of next-generation nanoparticle mediated multicomponent combination therapy and combination immunotherapy need to amplify the therapeutic potential by providing the enhanced stability of cancer drugs in biologically active form, reducing toxicity related issues, overcoming immunosuppression and preferentially accumulating at the tumor site. Due to lack of fenestered or discontinuous endothelium, effective delivery of clinically safe nanomedicine to less accessible tissues remains a considerable challenge. To achieve clinically potential, optimized nanoparticle designing, innovation in designing the mechanistically inspired nanoparticle, targeting specific metastatic foci, their delivery process and in vivo biodistribution based on structure and activity need detailed understanding. Integrating cancer biology and anti-metastatic nanotechnology, new strategies need to be engineered considering the biological mechanisms of various stages of metastasis. Tumor microenvironment specific targeted therapeutic intervention is desirable to improve the outcome. New guidelines need to be developed about engineering multicomponent combination therapy and it requires extensive studies on various classes of drug combinations, our understanding about inter-drug interactions for such cases, spatio-temporal release of anticancer drugs, nonspecific activation of the immune system by such multicomponent combination nanomedicine, diversity of metastatic foci, diversity of organ environment and delivery of biologically active multicomponent nanomedicine at the epicentre of solid tumor. Such therapeutics need to be developed by integrating clinical trials with an adequate number of patients having similar biomarker expression and the development of better pre-clinical models. Next-generation multifunctional cancer nanomedicine and nanoparticle mediated combination immunotherapy having mechanistically rational therapeutic combinations need to promote "multi-targeted therapy" by disrupting the adaptive chemoresistance and potentiating the effect of therapeutic combinations. Integrating proteomics data of patient samples, combination nanotherapy and immune-oncology, one may develop highly effective precision nanomedicine.

Competing Interests
The authors declare the following competing financial interests: We have filed patents on peptide based siRNA transporters (ap-

Author Contribution
Dr Kasturee Chakraborty, Archana Tripathi, Sukumar Mishra, Argha Mario Mallick and Dr Rituparna Sinha Roy designed and drafted this review article. All authors had read and approved the final manuscript.