**2.1 Tunable physicochemical attributes of nanomaterials compatible with biomedical applications**

Nanoparticles exhibit outstanding physicochemical attributes which can be manipulated to harness the best possible benefits out of them - their tunable diameter, high surface area, various morphologies, different concentrations and compositions, surface functionalization, etc. [15] (**Figure 1**). Interactions of NMs to the cell surface, their internalization, and subcellular localization, communication with the cells eventually contribute to therapeutic or adverse effects. Understanding the physicochemical attributes of NMs and their interactions with biological entities can help design superior NMs for further applications. We are jotting down the relevant physicochemical attributes of nanoparticles, which may modulate their function in therapeutic or toxicity aspects; thus, they need to be engineered wisely [16].

#### *2.1.1 Nanomaterials size*

For engineered nanoparticles, the primary crucial feature is their dimensions/size, which partially governs other physicochemical characteristics. The reduced diameter of the nanoparticles, provide possibilities for high cellular localization making them interact with cellular tissues, especially pathological tissue to a greater extent to attain specific biological outcome for the remedial purpose. Size-dependent bio-distribution studies were performed using three different sizes containing (20, 50, and 200 nm) drug conjugated silica nanoparticles. It revealed that nanoparticles having 50 nm diameter had the highest tumor localization, enhanced cancer tissue retention, and slower clearance [16]. Moreover, nano-sized particles preside over their pharmacokinetics, are predictable to traverse biological barriers, which is not possible for bulk

particles. Besides, ~50 nm diameter particles showed higher efficacy because of active engagement to the biological tissues, modulating pathways, and cellular activities [17].

#### *2.1.2 Nanomaterials surface charge*

The surface charge is a unique character of NMs to manage its therapeutic and toxicological effects and plays a significant role in electrostatic interactions of NMs and living entities (**Figure 1**) [10]. Besides, the cellular localization pathways and tissue interactions are regulated by the surface charge of the nanoparticles, thus playing a significant role in the compatibility and cellular toxicity. Several reports suggest that nanoparticles with a positive charge highly interact with the negatively charged cell membranes and provoke genotoxicity [18]. Positively charged cationic liposomal drug and gene delivery systems have been extensively studied for the last decade. It was recently shown that cationic lipoplexes are not showing any genotoxicological aberrations in the Swiss albino mice. Typically, cell membranes are anionic in nature; thus, negatively charged NMs have very slow cellular internalization compared to neutral and positive nanoparticles [14]. Surface potentials of metal particles in regulating different tumorous and non-tumorous tissue types are also established. Several studies have suggested the role of the surface potential of different nanoparticles and their interactions with the biological entities and how surface charge modulates their biological functions, which shed light to design and engineer nanoparticles for a selective cellular target for various diseases with minimal toxicity [16, 18].

#### *2.1.3 Surface functionalization*

Nanoparticles play a vital role in promoting intracellular delivery of encapsulated therapeutic agents and increase their retention in pathological tissues compared to healthy tissues [1]. Surface functionalization with suitable receptor-targeted ligands using different methods results in the formation of targeted nanoparticles with improved therapeutic response and minimized off-target side effects by prolonging their circulation time in blood, increasing target specificity, cellular uptake, and drug accumulation in the tumors (escaping lysosomal degradation and enhancing stimuli-responsive drug release) (**Figure 1**) [17, 19]. Depending on their application, nanoparticles are functionalized with different targeting ligands either by directly conjugating ligands to PEGylated nanoparticles through post-insertion technique or by covalent grafting on the surface of the nanoparticles. In this context, surface functionalization of nanoparticles with antibodies, peptides, folic acid, aptamers has been extensively studied. This prompts scientists to design and engineer nanoparticles for selective targeting and high retention in the tumor tissue rendering minimal toxicity to the vital organs [19, 20].
