**3. Mechanism of targeted drug delivery using nano-carrier**

Nanoparticles play a vital role in promoting intracellular delivery of enclosed therapeutic agents and increase their retention in the different pathological tissues compared to other therapies [21]. Like normal tissues, tumors need nourishments by means of food and oxygen and a capacity to remove metabolic excretes and carbon dioxide. Diverse patterns of tumor-associated neovascularization, obtained by angiogenesis, cope with these demands. Primary conservative treatment modalities

*Nanotechnology Application and Intellectual Property Right Prospects of Mammalian Cell... DOI: http://dx.doi.org/10.5772/intechopen.99146*

involved in cancer treatment are surgery, radiotherapy, and chemotherapy, while additional therapies such as immune therapy, targeted therapy, and hormone therapy are chosen depending on the type of tumor [22]. On the other hand, the failure of chemotherapeutic drugs to specifically target cancer tissue hinders many treatment modalities. It is habitually faster and economically cheaper to design an existing drug to encapsulate in a delivery system a more effective way to superior targeting of tissues than to invent a completely new one. The drug delivery mechanism can be classified into passive and active, respectively.

#### **3.1 Nanoparticle drug delivery by passive targeting**

Passive targeted drug delivery mainly depends on the physicochemical attributes of the NMs, such as shape, diameter, surface potentials, and pathophysiological conditions of the disease microenvironment. Intravenously injected drug encapsulated NMs tend to disperse throughout the body evenly [23]. However, unlike normal tissues, tumor cells tend to take up particles of a definite diameter to a greater extent than healthy cells due to the arrangement of capillary endothelial cells, accumulating extravasated molecules in the interstitial spaces poor lymphatic drainage increases the permeation and accumulation of drug-mediated NMs. This type of NMs accumulation in the tumor region is known as the EPR effect [1, 2]. The EPR effect is influenced by physicochemical attributes of NM including particle diameter, shape, and surface potentials greatly influence the circulation time, penetration speed, tumor localization, and intracellular internalization.

Particle diameter plays a critical role in achieving effective drug delivery as it enhances permeation and circulation time and reduces renal clearance. For example, phagocyte cells facilitate larger particle uptake, while non-phagocytic cells favor the uptake of smaller particles. PEGylated NPs reduced plasma protein adsorption on their surface and reduced hepatic filtration when their size is smaller than 100 nm [24]. Particle diameter with 20–200 nm effectively enhances the permeation in both hyper-permeable and poorly permeable tumors, and particles with less than 6 nm avoid renal clearance. The NPs surface potentials could play a vital role in circulation and cellular localization [24, 25]. NPs with positive surface potentials such as cationic liposomes induce non-specific interactions with blood components and aggregation of liposomes results in a reduction of EPR effect and increased renal clearance. However, positively charged NPs are more readily taken up by cancer cells. Whereas anionic and neutral surface potential-bearing NPs circulate longer in the blood circulation [1, 2, 24].

Besides, Polyethylene glycol (PEG) polymer is used as a stabilizer (stealth liposomes) that increases the circulation time in blood up to 24–48 hours and improves *in vivo* stability [26]. PEG-coated liposomes induce the 'steric stabilization effect' by creating hydrophilicity on the surface of liposomes that shield surface charge and increases the repulsive forces between liposomes and blood components. Thus, it prevents aggregation of liposomes and opsonization by the reticuloendothelial system, macrophages, mononuclear phagocytic cells and prolongs their systemic circulation. On the other hand, PEG-coated liposomes induce PEG-specific IgM antibodies, enhancing hepatic uptake and rapid clearance of liposomes from systemic circulation on subsequent administration. PEG corona produces steric hindrance with tumor cells that prevent effective internalization, which could be minimized by using short PEG chains with molecular weight less than 1000 Da or by designing PEG with enzyme-cleavable bound or tumor-targeting ligands [20, 26].

To investigate the influence of shape on the cellular localization of NPs, Li et al. conducted large-scale molecular simulations to evaluate different NP geometries with identical surface area, ligand-receptor interaction strength, and PEG grafting density. They observed that spheres exhibited the fastest internalization rate, followed by cubes, while rods and disks were the slowest. Many liposomal formulations have received clinical approval, like Doxil, Abraxane, etc. However, nanoparticles grafted with PEG prolong the systemic circulation of the particles and induces the EPR effect in tumor cells, but lack of target specificity often results in reduced therapeutic efficacy [27]. Because of that, more than 95% of passively targeted formulations fail to go bench to bedside.

#### **3.2 Nanoparticle-based drug delivery by active targeting**

An ideal nanoparticle delivery system should be proficient at reaching, recognizing, and delivering its payload to determined morbid tissues and avoid druginduced toxicity to healthy tissues [7]. Therefore, functionalizing specific targeting moieties on the surface of nanoparticles is the most usual plan. Nanoparticles are functionalized on their outer surface by targeting moieties such as small molecule ligands, monoclonal antibodies, aptamers, cell-penetrating peptides, and proteins that are internalized into morbid cells by interacting with cell surface receptors like folate receptors, transferrin receptors, tyrosine kinases like EGFR, and so on [28] (**Figure 1**). Cell surface receptors that are significantly overexpressed in diseased cells, compared to normal healthy cells, provide a potential target for the design and development of actively targeted drug delivery and help to reduce off-target effects [7, 17]. These ligand moieties can interact with target-specific diseased cells and protect nanoparticles from enzymatic demolition.

Targeted drug delivery significantly minimizes the toxicity and induces patient compliance with less frequent dosing. Active targeting depends on ligands bound to the NP surface to improve their uptake selectivity and protect NPS from enzymatic destruction. The main principle of active targeting involves functionalizing an NP with a ligand that binds to a molecule overexpressed on cells. Ligands with a high binding affinity to a specific cell type exhibit higher delivery efficiency. One important thing to consider is that healthy cells still express the same molecule, and as healthy cells greatly outnumber, the chances of NPs missing their target will also increase. An intelligent selection and functionalization with multiple ligands can effectively mitigate the problem. Apart from this, active targeting mainly determined the kind of nanoparticle carrier, ligand targeting specific receptors, functional agents used for linking a ligand to the nanoparticles, hydrophilic polymers, and encapsulated active ingredients [28, 29].

Targeting tumor cell surface receptors is a common approach in active targeting. Nanoparticles were linked with targeted ligands for targeting specific cell receptors and thus upregulated the intracellular localization and therapeutic efficiency. Liposomes are conjugated with antibodies, a Y-shaped glycoprotein, or its fragments often termed as immunoliposomes, increasing the specificity of liposomes by targeting antigen-presenting cancer cells, which undergo endocytosis and destroy cancer cells followed by immune system clearance [28]. Folate receptors are membrane proteins overexpressed by various tumor cells. Folic acid is a ligand for targeting folate receptors, which pose high affinity, stability, and conjugation capacity [30]. It is conjugated with nanoparticles and a PEG spacer that inhibits steric hindrance between the cells and liposomes, which helps to increase

### *Nanotechnology Application and Intellectual Property Right Prospects of Mammalian Cell... DOI: http://dx.doi.org/10.5772/intechopen.99146*

cellular uptake and drug delivery of folate-targeted anticancer drugs. Targeting folate receptors with folic acid ligands helps deliver therapeutic and imaging agents effectively to the requisite site. Endothelial growth factor receptors (EGFR) overexpressed in solid tumors like non-small cell lung cancer, colorectal, squamous cell carcinoma of the ovary, kidney, head, neck, pancreas, prostate, and breast cancers can help in designing EGFR targeted drug delivery system. Antibody fragments used for targeting EGFR are functionalized on nanoparticle surfaces in order to acquire high targeting specificity [31]. Fibroblast growth factor receptors are overexpressed in cancers like lung, prostate, bladder, etc. Several groups have reported remarkable interaction of FGFs conjugated liposome with FGFR and discussed in detail [32, 33]. Overexpression of CD44 is observed in cancers like leukemia, ovarian, colon, gastric, pancreatic, and epithelial cancers. Hyaluronic acid acts as a ligand for CD44 and is used to deliver gemcitabine and DOX encapsulated within the liposomes [34].

Targeting the tumor microenvironment is another approach in active targeting, and one aspect is targeting the tumor vasculature instead of the tumor. This approach helps in the targeted destruction of neo-angiogenic blood vessels essential for tumor growth and metastasis [29, 35]. Vascular endothelial growth factor receptors (VEGFR) play a significant role in tumor angiogenesis and vascular permeability and regulate other aspects of tumorigenesis. Bevacizumab, a monoclonal antibody approved by USFDA, is used as an anti-human VEGF for targeting VEGFRs and FGFRs tyrosine receptors for active targeting [29]. Vascular cell adhesion molecules (VCAM-1) are cell adhesion molecules (CAMs) present on the endothelial cells responsible for inflammation. VCAM-1 is overexpressed in cancers like non-small cell lung cancer and tumor vasculature. Anti-VCAM and Fab-conjugated liposomes have high cellular uptake into Human Umbilical Vein and Endothelial Cells (HUVEC) compared to conventional liposomes [36].

Matrix metalloproteases (MMPs) are calcium-dependent endopeptidases involved in remodeling extracellular matrix, tumor invasiveness, and metastasis by modulating the formation of new blood vessels [37]. Conjugating MMP-2 cleavable peptides to liposomes loaded with cell-penetrating peptides increase the tumor selectivity. αβ-integrins are the heterodimeric transmembrane glycoproteins that facilitate the adhesion of endothelial cells with adjacent tissue and blood vessels. A tripeptide Arg-Gly-Asp (RGD) exhibited high specificity for αvβ3 integrin helps in developing integrin targeted liposomes, which inhibits adhesion and angiogenesis in the tumor microenvironment (TME) [38]. Active targeting amends the intuitive patterns of a nanocarrier, directing to the specificity of the pathological tissue. In contrast, passive targeting delivery depends on the natural distribution of the therapeutic motifs and the EPR effect. Both the targeting mechanisms depend on blood circulation and the location of initial drug delivery. However, rare commercial advances are made using actively targeted NPs [39].
