Design Considerations for Chemotherapeutic Drug Nanocarriers

The drugs used in conventional chemotherapy targets both cancerous cells and non-cancerous cells. This makes the treatment of the cancer cells highly ineffective due to excessive toxicities [1]. Various attempts have been made to combat tumors specifically to spare non-cancerous cells [2]. But, cancer cells develop resistance to the conventional chemotherapeutics and the newer molecular approach thereby evading the cytotoxicity [3]. Due to several advantages, nanomedicines can be a promising approach for an effective and specific chemotherapy. Firstly, due to high surface to volume ratio, nanoscale carriers reduce the distribution volume of the drug [4,5], therefore improving the pharmacokinetics and the biodistribution of the drug to specific organs [6-9]. Secondly, specificity imposed to the nanocarriers Abstract


Introduction
The drugs used in conventional chemotherapy targets both cancerous cells and non-cancerous cells. This makes the treatment of the cancer cells highly ineffective due to excessive toxicities [1]. Various attempts have been made to combat tumors specifically to spare non-cancerous cells [2]. But, cancer cells develop resistance to the conventional chemotherapeutics and the newer molecular approach thereby evading the cytotoxicity [3]. Due to several advantages, nanomedicines can be a promising approach for an effective and specific chemotherapy. Firstly, due to high surface to volume ratio, nanoscale carriers reduce the distribution volume of the drug [4,5], therefore improving the pharmacokinetics and the biodistribution of the drug to specific organs [6][7][8][9]. Secondly, specificity imposed to the nanocarriers

Abstract
The use of nanotechnology in delivering the chemotherapeutics drug has gained much attention recently. It is capable of killing the cancer much more effectively than any other method. The drug delivery systems using nanocarrier significantly enhances the efficacy of drug by improving the pharmacokinetics and the distribution of the drug to specific organs. For designing an effective nanocarrier, an insight of size, shape, surface chemistry and geometry is important. This review gives a map of guidelines for design of nanoparticle based chemotherapy. It reviews the mechanism of delivery in different pathways, physiology and chemistries involved and barriers to transport and delivery of nanocarrier based drugs, specifically for chemotherapeutic drugs. The microenvironment and physiology of a tumor site and its chemical environment is also reviewed, focusing on the impact on delivery. This review is an attempt to map the parameters that will help effective design of nanoparticles as drug carriers for chemotherapeutics. It discusses the accurate designing of nanocarriers as well as the effect of the environment to which a nanocarrier is exposed inside the body, its fate and uptake.
lowers the cytotoxicity to healthy tissues [10]. Thirdly, easier delivery of hydrophobic drugs in parenteral mode [10][11][12]. Fourthly, the stability of several therapeutic drugs like peptides, hydrophobic compounds, etc. is found to increase using this delivery system [13][14][15]. Finally, safe nanocarriers due to biodegradable polymers due to lower side effects and better efficacy [16][17][18]. Figure 1 illustrates the different advantages offered by nanoparticles based drug delivery.

Scope of the Review
To design an effective nanocarrier, it is important to understand the environment in which a nanocarrier will travel its fate and challenges at different steps. Nanocarriers can only designed correctly with enough information about delivery pathway. Different pathways offer different challenges to a naoncarrier. These challenges can be overcome by considering all important factors responsible to its movement, functionality, recognition, specificity, etc. This review assesses all of these routes and environments to which the carrier is exposed and the barriers in each of these pathways. Moreover, this review acts as a guidelines and a map for the basic and essential parameters for designing nanocarriers for cancer therapy. This review will discuss all important factors for an effective nanocarrier design and help engineering the the nanoparticles in a way to achieve maximum uptake, minimum clearance by reticulo-endothelial system (RES), maximum transport in tumors and controlled release of drugs to constitute an efficient drug delivery system.

Drug Delivery Systems Transdermal drug delivery system
In this approach, the human skin is used as the primary route of administration of drugs into the bloodstream. Bioactive compounds are applied on to the skin to achieve therapeutic blood levels for treatment of diseases which are distant from the site of application. Human skin surface provides a surface area of approximately 2 m 2 with 1/3 rd of blood supply of the body. It is one of the most conventional approaches for several decades. Drugs administered through this technique have to pass all the skin barriers and enter into the systemic circulation, which can be achieved by two ways: i. Transcellular pathway, in which a drug passes through phospholipid membranes and the cytoplasm of the dead keratinocytes, which forms the outermost layer of epidermis (stratum corneum), ii. Intercellular pathway, where a drug finds its way within the small spaces between the cells of the skin.
Despite being easiest mode of delivery, owing to convenience, absence of any complications (like those that affect delivery through gastrointestinal (GI) tract) and reduced side effects, this approach suffers from several disadvantages like local irritation, edema, low permeability of skin, uncontrolled release of drugs [18][19][20][21]. Barriers to transport of drugs through the skin limit the volume of drug that can be transported for successful administration of therapeutics.

Parenteral drug delivery system
Parenteral route of administration refers to injection, infusion or implantation of drug into the human or animal body. It can also be called as injectable drug delivery, which can be subcutaneous (SC/ SQ), intramuscular (IM) or intravascular (IV). Drugs with poor bioavailability and low therapeutic index can be delivered using this method. It has been reported that parenteral drug delivery market constitutes one of the largest segments and accounts for nearly 30% of the total market share. Immediate physiological response, improved bioavailability of drugs, the absence of GI tract complications (which includes drug-degradation), rapid and maximum absorption, flexibility are some of the major advantages for parenteral delivery system [22][23][24][25][26][27]. Some major disadvantages are higher cost of manufacturing; invasive, aseptic conditions need to be followed. Trained healthcare professionals are required. These factors further add to the cost of this route for therapeutics delivery. In addition, there are other barriers. Drugs once injected cannot be removed from bloodstream. Patients feel pain or discomfort during injection, and this often results in poor patient compliance and acceptability especially if multiple daily injections required like in case of insulin, etc. [28,29].

Transmucosal drug delivery system
Transmucosal routes of delivery involve drug administration through mucosal linings of nasal, rectal, vaginal, ocular, and oral cavity. Mucosal linings are highly vascularized, have rich blood supply and good permeability. It provides several advantages over injectables and enteric routes. The major advantages of using mucosal route are the bypassing of GI tract and first-pass metabolism in liver. Drugs which are absorbed enter directly into the bloodstream and hence reducing the GI tract complications [29]. Due to high accessibility, oral mucosa has also been found to be the most acceptable route of administration.
The hurdles in therapeutics delivery using this route include high enzymatic environment of oral mucosa. The carrier / drug system needs to be permeable through barriers of oral mucosa. In some cases saliva (or other secretions) wash away the drug; there is a need for high mucoadhesion for effective delivery.

Oral drug delivery system
The oral route is considered to be the most widely accepted mode for drug delivery owing to the convenience, ease of administration and cost effectiveness [29,30]. This mode of administration of drug relies on the absorptive capacity of the gastrointestinal (GI) tract. The drug administered orally must overcome the acidic environment and enzymes present in GI tract. Hence, drug delivery vehicles are needed to increase the oral absorption, easy passage through intestinal membrane and avoid the destructive nature of GI tract [31]. This is accomplished with the use of nanotechnology which enables (i) the delivery of poorly water-soluble drugs, (ii) the targeting of drugs to the specific regions of the GI tract, (iii) transcytosis of drugs across the intestinal barriers, and (iv) intracellular delivery of drugs [32]. Use of nanomedicines is highly advantageous as apart from increasing the efficacy and tolerability of drug it provides wide range of nanosystems for oral drug delivery [33,34]. Nanocarriers ranging from polymeric nanoparticles, solid lipid nanoparticles, nanocrystals and self-nanoemulsifying systems have been applied for oral drug delivery [35].

Targeted drug delivery
Targeted drug delivery is the ability to direct any therapeutic agent to desired site of action specifically, with little or no interaction with non-target cells/tissues. "Clever" delivery system includes the parallel behavior of three components: the targeting moiety, the carrier and the therapeutic drug. Drug-targeting can be an (i) active strategy, which is also referred as receptor-ligand or ligand based targeting or the (ii) passive or physical targeting, which introduces the drug carrier complex into the body that can avoid elimination from body's defense mechanism, retains itself in circulation and reaches to the target site [36].

Reticuloendothelial system (RES)
The reticuloendothelial system (RES) is a physiological system involves in the elimination of foreign macromolecules and particles from the body. It is a part of the immune system that includes macrophages and monocytes. Such cells have the ability to take up particles and dyes through phagocytosis, a process involving the engulfment of solid particles by the cell membrane (also known as "cell eating"). RES functions to remove the dead cells from the circulation and to introduce phagocytic cells for inflammatory and immune responses. Different forms of drug carriers like liposomes, emulsions, nanocomposition, bilayer structures when administered intravenously are found to be restricted by the organs of RES (liver, spleen, bone marrow) [37-39].

Tumor Microenvironment
A detailed study of the tumor microenvironment is necessary for designing the effective delivery technique for chemotherapeutic drugs. Cancer cells exhibit a different microenvironment in comparison with the normal cells, such as, vascular abnormalities, oxygenation, perfusion, pH and metabolic states. Hence a better understanding of the tumor vasculature and interstitium help researchers to develop different therapeutic strategies. Tumor cells exhibit abnormalities in blood vessels, lymphatic system, vascular barrier, interstitium. Due to angiogenesis, growth of new cells occur from pre-existing ones which leads to highly dilated with wide interendothelial junctions, large number of fenestrations and transendothelial channels formed by vesicles, thick basement membrane, and leaky vessel walls with high permeability [40][41][42][43][44]. This abnormal growth helps tumors obtain extra oxygen and nutrients necessary for their growth and proliferation. All these abnormalities help molecules to transit across tumor vessels by phenomena called as enhanced permeation and retention effect (EPR) ( Figure 2).

Drug Nanocarriers
Drug carriers are vehicles for protected transport of drugs to affected sites and their controlled release in the body. Therefore, the size and shape of the particles as well as their surface functionality should be manipulated in such manner which facilitates their transport through barriers of different membranes and tissues as well as the protection of the encapsulated drug during transport. Nanocarrier based drug delivery strategies leverage multiple aspects of nanoparticle structures: (i) nanomaterials provides large surface to volume ratio in comparison to other conventional drug vectors hence imparts them with properties like specificity, selectivity, versatility, etc.(ii) nanosize allows transportation of drugs through cells and membranes, and (iii) nanosize enables drugs to avoid RES. Dendrimers, polymeric micelles, polymeric nanoparticles, viral nanoparticles, liposomes are some the nanocarriers which have been used in the past for studying their applications in the field of cancer drug delivery.

Design Parameters for Nanocarriers
It is important to understand the interactions between the nanostructure and a biological membrane, before designing a nanocarrier. Past studies focussed on developing novel nanomaterials but the designing properties like nanostructures, size, shape, and surface chemistry did not get much attention For example, in delivery of any cancer drug to tumors, size, shape, surface charges and chemistry of nanocarrier influences delivery efficiency, and drug distribution. This insight can be used to redesign the nanomaterials accordingly so that large fraction of nanocarriers can penetrate and accumulate inside tumors. Moreover, it has been recently reported by Albanese et al. that even the interactions between the ligands on nanoparticles surface and the receptors present on the cell surface are also dependent on the engineered geometry of nanoparticle. Therefore, there are certain points ( Figure 3) which should be kept in consideration while engineering the nanocarrier. Such as: • It should escape clearance mechanism.
• It should be in circulation.
• It should overcome drug resistance.
• It should have appropriate charge to adhere to the cell membrane.
• It should have proper ligands to bind with the receptors.
• It should be in a size small enough to escape phagocytosis and large enough to escape translocation in tissues and organs.

Surface charge
Nanoparticle properties for therapeutic applications are governed by several factors such size and shape, surface charge of the nanoparticles. One of the most important properties of nanoparticle to be controlled in the nanoparticle design is the cytotoxicity of nanoparticle. Charge density and charge polarity plays a major role in the cytotoxic action of a nanoparticle. Studies have shown that charged nanoparticles are more cytotoxic than neutral charged nanoparticles

Nanoparticle shape and geometry
Apart from the various factors discussed, particle shape also contributes to the property of nanoparticles. Nanoparticle shape is a critical factor in drug delivery. There are several evidences that show the importance of particle shape on the release of drug. Studies have shown the controlled release of drugs is possible with the use of hemispherical sized particle, but not if the size of the particle is in the millimeter range [71]. Non-spherical particles show different rates of degradation because of different areas of thickness [72]. Geng et al. [73] found a positive correlation between in vivo blood circulation of nanoparticle and length-width ratio of the nanoparticle.
Transport of the nanoparticle will be greatly affected by the shape of the nanoparticle. Movement of the particle is dependent on the symmentry of the particle. Non-spherical particles may tumble when flowing through the organs such as liver and spleen or when the particles are encountered by the obstacles in the blood vessels [74].
Another factor governed by the particle shape is the targeting ability of the particle. Apart from the surface area of the particle, curvature, opsonin adsorption also affects the ligand targeting by the particle. Once the particles get attached to the contours of target plasma membrane, the protruding ends of particle are detached by the flow of blood. Thus, the protruding ends of the particle determine the longevity of the targeted attachment [75]. Particle shape not only determines the internalization of the targeted particles but also the transport and sorting of the particles once inside the cell [76].
Methods to fabricate non-spherical nanoparticle: Particle shape has not been investigated in detail particularly because of the limited methods available for the synthesis of non-spherical nanoparticle [75]. In recent years, several methods have been designed to fabricate the non-spherical nanoparticles, out of which the two main methods are: 1) synthesis of non-spherical nanoparticle from the beginning; 2) Alterations in the spherical particles fabricated earlier into nonspherical particle. Synthesis method involves the use of techniques such as lithography, microfluidics and photopolymerization [77,78].
The second method involves the manipulation of fabricated spherical particles into non-spherical particles. Studies have shown the formation of polystyrene sphere particles because of the self-assembled polystyrene spheres on the surface of a droplet [79]. Inspite of the advantages of the methods of fabrication of non-spherical nanoparticle, there are some limitations also. The most important limitation is the shape produced in the methods. For example, microfluidic methods generate two dimensional shapes and microchannel geometry is one of the limitations of this method [77] (Figure 4).

Surface chemistry and modification
Surface chemistry dictates the fate of a nanoparticle during clearance or uptake in circulation. It is essential for nanoparticles to have long circulation half life and to escape from macrophages ( Figure  5). Therefore, residence time or circulation time is an important factor for effective designing of a nanocarrier. In cancer therapy, long circulation is required for passive targeting because EPR effect is observed in tumor vasculature after multiple passes [80][81][82][83]. But to achieve this, nanoparticles should be made such that drug degradation can be avoided. Therefore, surface modification is required to make the nanoparticle more effective in carrying the loaded drug to the targeted site. Nicholas et al. [84] reported that blood half-life of nanoparticles is dependent on the surface hydrophobicity of nanoparticles. Nanoparticle's surface hydrophobicity determines the amount of proteins (opsonins) adsorbed on the surface. Particles which are more hydrophobic suffer more opsonization. Past studies have reported the PEG-ylation of the nanoparticles as hydrophilic blocks [83,85,86]. It increases the circulation time by escaping through immune cells (opsonisation). Past studies reported that PEG (Polyethylene glycol) prevents aggregation of the nanoparticles, helps in stabilising the nanoparticles, providing a neutral surface charge to nanoparticles, nanoparticles, escape from clearance by preventing from opsonins [87]. For effective modification of the surface, length and density of the PEG plays vital role [88,89]. PEG shields the inner core of nanoparticle from blood proteins by  forming a brush layer on the surface of nanoparticles. The access of encapsulated drug is restricted to the enzymes by modification of the nanoparticle surface therefore, improving pharmacokinetic profile and reducing non-specific toxicity [89][90][91][92][93][94][95][96][97][98][99][100][101][102][103][104][105].

Effect of size
Size of a particle influences the functionality of that particle like its uptake, residence in circulation, adherence, degradation as well as clearance [110][111][112][113][114] Size governs the movement of the nanoparticles inside the tissues. Figure 6 represents the effect of size on nanoparticles drug delivery. Champion et al. [75] reported that the movement of the particles inside tissues is dependent on the size as their movement can be sterically hindered in extra-cellular matrix. Based on the relationship between particle size and its curvature (for spheres), size of the nanoparticles along with surface chemistry, may also affect opsonization [115][116][117][118][119][120][121]. Recently, it was reported that [121] reported that size also play vital role in targeting nanoparticles accumulate inside the tumors by EPR effect, which in turn depends on the extravastion through the gaps in tumor vasculature. The ideal size range reviewed in past studies is 50-150 nm. However, a study reports that ultra-small gold nanoparticles of size range ≤ 10 nm exhibits uniform distribution inside tumor tissues due to their ability to diffuse through tissues [122]. Fang et al. [123] carried out a study with PEG-PHDCA nanoparticles of size range 80-240 nm for cellular uptake and it was reported that smaller nanoparticles shown better circulation and accumulation but uptake was poor.
Particle diameter and size can be controlled by varying different   [117] have shown the effect of particle size on the degradation. There is no direct relationship between the initial degradation rate and size of the nanoparticles and microparticles. The size and diameter of a particle guides its way inside a bloodstream, diffusion in cells or membranes, air-passage or gastro-intestinal tract [124]. Size is an important factor to decide the destination and fate of the nanoparticles inside the body. Illum et al. [115] and Tabata and Ikada [116] reported the fate of the particles inside body. Tables 1-5 shows the effect of size and their fate inside of body.
Designing shape and size specific nanocarriers: Previous researches over several decades focused on designing of nanocarriers by two major approaches-bottom-up synthesis and top-down approach. Designing liposomal carriers, micelles, polymeric nanospheres, drug encapsulated polymeric nanoparticles are some vehicle which fall under "bottom up" category. This approach is based on self assembly and emulsion systems. Major advancement has been made recently in fabrication technology by introducing "top-down" approach in micro and nano-fabrication system using electromechanical approach (MEMS & NEMS). They have exhibited the potential for designing nanoparticles with precision in particle shape and size. Such approach can provide control over particle size, functionality, particle geometry with accurate precision. This approach can also have ability to resolve the limitation of bottom-up approach.

Bottom-up synthesis:
This approach has been extensively studied in past and several types of potential nanocarriers have been developed using this method for example, polymeric nanoparticles, micelles, liposomes, nanoemulsions, dendrimers, biodegaradble and nonbiodegradable carriers, solid lipid nanoparticles, magnetic nanoparticles etc. Each of these carriers has been extensively reviewed by various researchers in last decade. Several invitro and invivo studies have been done and are still going on. Majority of these carriers are colloidal systems which are governed by different forces like hydrophobic interactions, vander-walls forces, hydrogen bonding, and ionic interactions. Often, high polydispersity have been exhibited by such system. Such systems sometimes undergo certain limitations. Invivo drug release profiles, physicochemical characteristics, degradation kinetics of these carriers are difficult to evaluate and reproduce as they are variable.

Top-down synthesis:
Recent advancements in designing of nanoparticles have been made by micro-and nanofabrication techniques [123,124]. Different nano imprint lithography processes fall under this category (Figure 7). Today, advance researches in the field of nanofabrication for drug delivery are going on using soft lithography [125], thermal embossing [126][127][128][129], step and flash lithography [129,130], and UV embossing [131][132][133]. This technique has already been explored by Shvartsman and Desai et al. [134][135][136]    • Facilitated by catalyst. [104] Click chemistry • Specific conjugation at desired location (due to azide & alkyne reactive groups) • Useful where orientation & stability of moiety is important. [105] Linker chemistry Linker provides a control over molecular orientation and useful for controlled delivery systems [106] Electrostatic interactions Cationic-anionic interactions [107] Hydrophilic/hydrophobic interactions Nanoparticle's surface engineered with hydrophobic surface which can adsorb hydrophobic drugs. [106] Affinity interactions Surface modified with streptavidin for specific bioconjugation.

Conclusion
This review explains the parameters necessary for nanocarrier design to combat tumors. This review specifically focuses on challenges in a perfect nanocarrier development. There are conflicting effects of size or surface functionality in transport through membranes, blood stream and cellular uptake, for example, and this leads to a design sweet spot that allows for efficacious delivery. It describes the role of various aspects of the nanoparticle in supporting and enhancing drug delivery. This review develops a map for design of nanoparticle based chemotherapeutic strategies by recognizing the mechanisms of transport in the delivery pathway of choice, the barriers to these transport mechanisms, and the role of structure, functionality and material of nanoparticles in inhibiting or supporting transport.