Effects of Size and Surface Charge of Polymeric Nanoparticles on in Vitro and in Vivo Applications

Biodegradable polymeric materials are the most common carriers for use in drug delivery systems. With this trend, newer drug delivery systems using targeted and controlled release polymeric nanoparticles (NPs) are being developed to manipulate their navigation in complex in vivo environment. However, a clear understanding of the interactions between biological systems and these nanoparticulates is still unexplored. Different studies have been performed to correlate the physicochemical properties of polymeric NPs with the biological responses. Size and surface charge are the two fundamental physicochemical properties that provide a key direction to design an effective NP formulation. In this critical review, our goal is to provide a brief overview on the influences of size and surface charge of different polymeric NPs in vitro and to highlight the challenges involved with in vivo trials.


Introduction
Manufacturing effective drug delivery system is a critical challenge in nanomedicine since nanocarriers are expected to reach and accumulate in the site of interest.As a consequence, numerous drug delivery systems have been investigated in vitro and in vivo to deliver a wide range of drugs and molecules.To conquer the challenge, with the aim of avoiding uncontrolled biodistribution, rapid clearance and systemic toxicities in healthy tissues polymeric nanoparticles (NPs) have gained higher interest among all the novel formulations.Research during the past few decades proves their beneficial features in formulation design, characterization, behavior and application [1].The in vitro and in vivo fate of NPs is particularly depended on uniformity of particle size and zeta potential.Change in these properties has significant biological implications on cellular internalization, pharmacokinetics, and bio-distribution [2].These characteristics of NPs facilitate the opportunities for therapeutic application, which can be confirmed by in vitro and in vivo studies [3].The aim of most nano-devices is to prevent the degradation of drug followed by higher bioavailability in cellular level and to regulate its pharmacodynamics profile.Thus, the nanomedicine platforms could serve as a drug delivery system that is able to transport a high dose of therapeutics selectively to the desired site of action.Although very few investigations have been performed, most of the articles related to exploring the effects of size and surface charge of NPs have been discussed in this review.This review provides details on the fate of different polymeric NPs and will discuss how the size and surface charge of polymeric NPs are involved in desired effects for both in vitro and in vivo applications.Moreover, other polymeric NPs using various preparation methods have been also summarized in Table 1, which could be considered for further size and surface charge related experiments.

Polymeric NPs
For an ideal drug delivery system, recognition of the polymer's potentiality has been evaluated since 1960's [4].
Over the past few decades, two main classifications of polymers have been discovered as synthetic and natural, each with various types and sub-types.Synthetic polymers are chemically synthesized based on repeated structural units, whereas natural polymers are obtained from natural sources.Primarily two types of polymeric NPs have been developed for drug delivery purposes i.e. nanocapsules, in which a core of encapsulated drug is surrounded by polymeric membrane or shell; and nanospheres, where drug is distributed/adsorbed throughout a matrix [5].The most important feature of polymers is the degree of biodegradability, which is an important criterion to differentiate some slowly biodegradable polymers such as polystyrene (PS), poly (cyanoacrelates) (PCA), polyethylenimine (PEI) and poly (methyl methacrylate) (PMMA) [6]- [11].On the other hand, some synthetic polymers such as poly (ɛ-caprolactone) (PCL), poly (lactide) (PL), poly (glycolide) (PGA), poly (D, L-lactide-co-glycolide) (PLGA), and some non-synthetic polymers (e.g.chitosan) are categorized as readily biodegradable materials [12]- [16].Polymeric NPs are capable to maintain high stability in systemic circulation with enhanced half-life, which can be further optimized by controlling the release of therapeutic agents from the NPs.Moreover, polymeric molecules have various solubility profiles in wide range of solvents.This is advantageous for surface modification or functionalization to achieve different purposes of delivery and targeting.Subsequently, both doses and frequency of administration of therapeutic agents can be reduced due to high payloads into nanocarriers, leading to superior efficacy and minimizing the side effects.Besides, polymeric NPs of desired physicochemical properties are capable of preserving their content from hepatic metabolism, enzymatic degradation and rapid clearance.Specifically, the enormous surface area of polymeric NPs is an attractive feature to control the release kinetics, drug loading capacity and administration route, which can regulate the fate of drug into the body [17].However, only few of them have been approved by health regulatory agencies for human trial to apply for carrying a wide range of diagnostic and therapeutic agents to the desired site of action [18].

Effect of Particle Size and Surface Charge Based on in Vitro Studies
Different types of NPs have been widely applied as drug delivery vehicles for diagnostic and targeted therapy (active or passive) to achieve maximum cellular uptake and therapeutic bioavailability [19] [20].Continuous physicochemical changes in the development of polymeric NPs may have substantial implications in the cellular internalization and biological processes [21].The experiments performed to evaluate the influence of particle size and surface charges of NPs are expected to explain how these physicochemical properties influence the cell uptake through various pathways towards optimum biodistribution.Cellular internalization or uptake is the most important physicochemical criteria prior to in vivo application.Uptake of small molecules by any cells depends mainly on endocytosis among all other mechanisms (Figure 1).Endocytosis is the bulk active transport process through lipid bilayer wrapping using energy in the form of ATP to form required vesicles.Two main endocytosis mechanisms are reported as phagocytosis and pinocytosis [22].Phagocytic cells (e.g.macrophages, neutrophils, dendritic cells, etc.) mediated cellular internalization is mostly involved with engulfing the large particles (>1 µm) [23].Adsorption or receptor dependent internalization is the main mechanism of pinocytosis, which is mainly related to particle uptake by the cells through different pathways such as macro-pinocytosis, clathrin mediated, caveolin dependent or independent pinocytosis [3].Size and surface charge of polymeric NPs are likely the preliminary physicochemical variables, which govern the endocytosis dependent cellular uptake.Besides, positive charge of the surface of polymeric NPs may endorse more cellular attachment causing higher uptake either by endocytosis or by direct penetration, since cationic surface of polymeric NPs interacts with anionic terminal of phospholipid, proteins and glycans on the cell surface due to the electrostatic interactions [23].
An interesting experiment by Bhattacharjee et al. demonstrated the effects of size and surface charge of fluorescent, monodisperse tri-block co-polymeric NPs based on cellular uptake through different endocytotic pathways [24].They synthesized polymeric NPs (PNPs) with two different sizes (45 and 90 nm) and surface charges such as neutral (PNP-OH, −4 mV), positive (PNP-NH 2 , +22 mV) and negative (PNP-COOH, −19 mV) to observe the in vitro cellular uptake into NR8383 (rat macrophage) and Caco-2 (human colonic adenocarcinoma) cells.For size dependent cellular uptake, a relative uptake study was carried out, which revealed the higher intracellular uptake by positively charged polymeric NPs with lower size compared to the other formulations.Inhibition of endocytic pathways was adopted to observe the role of endocytosis based cellular internalization of polymeric NPs tracked by two mechanisms such as decreasing temperature to 4˚C of experimental unit and exposing cells with 2-deoxyglucose (2-dOG) and sodium azide (NaN 3 ).Both inhibitory approaches showed considerably lower uptake, which proved the higher uptake by positive charged polymeric NPs compared to that of neutral to more negative charged polymeric NPs.Followed by the same strategy to block the clathrin and caveolin mediated endocytosis, cells were exposed with hypertonic 450 mM sucrose solution and methyl-beta-cyclodextran, respectively.Meeting the claimed fenestration sizes of these receptors dependent endocytosis, both inhibitions resulted with reduced uptake with smaller size after treating these cells with polymeric NPs, however the uptake was varied with charge variations.For clathrin dependent endocytosis, uptake by both neutral and negatively charged polymeric NPs was higher (65% and 75%, respectively) than positively charged polymeric NPs (less than 38%), however an opposite result was found for caveolin dependent endocytosis.
On the other hand, Lai et al. investigated that polystyrene (PS) NPs with smaller size (>42 nm) were successfully internalized into HeLa cells following clathrin and caveolin independent endocytic pathways avoiding endosomal or lysosomal accumulation [25].Recent studies revealed that positively charged NPs uptake was related to energy dependent process such as proteins dynamin and F-actin but negatively charged NPs were not dependent on dynamin proteins around the cell membrane [26].Moreover, highly positively-charged NPs could cause perforations in the cellular lipid bilayer to enter the cells by-passing endocytic pathways [23].
Another in vitro study for both fluorescent PS NPs and Coumarin-6 NPs in Caco-2 cells by Win et al. was performed to assess the effects of different polymeric NPs size [27].Raw Coumarin-6 could not increase the cell uptake, however fluorescent PS NPs of 100 nm to 200 nm size showed the highest percentage of uptake.Smaller particles (50 nm) showed the lowest uptake and particles as large as 1000 nm showed decrease in uptake, which could be attributed to the uptake by other cellular mechanisms.
Optimization of antigen delivery to human dendritic cells (DCs; antigen presenting cells) is a challenge for advanced vaccine delivery systems.To identify the effects of particle size and surface charge on human DCs, in vitro cell uptake study has been investigated by Foged et al. in 2005 [28].They designed the experiment based on wide size ranges (0.1 µm to 4.5 µm) of fluorescent PS NPs with different surface charges (+12.4 to −66.9 mV) after surface modification.Flow cytometric analysis of DCs after 24 hour incubation showed that lower percentage of DCs had taken up 4.5 µm particles (30%); whereas the highest cellular uptake (60%) was ob- served for 0.5 µm and 0.1 µm sized particles.To optimize the charge dependent interactions, particles with two sizes (0.1 and 1 µm) were modified by attaching variety poly amino acids/proteins covalently utilizing surface amine and carboxyl groups.Sterically same positive and neutral charges particles were obtained using polypeptides poly-l-lysine (PLL) and poly-d-l-alanine (PA), respectively.After 24 hours incubation, only 10% cellular uptake was observed with negatively charged 1 µm size particles, whereas positive charged particles were accounted for 60% uptake.However, around 90% uptake was observed for lower size (0.1 µm) particles with positive charge.
Prior to in vivo administration, it is essential to consider the compatibility, safety and biodegradability of the particles with the human blood and cells.To investigate the efficiency of particle size and surface charge in in vitro cellular uptake and blood compatibility, recently Dash et al. employed chitosan/polyglutamic acid hollow spheres to treat human umbilical vein endothelial cells (HUVECs) and human umbilical artery smooth muscle cells (HUASMCs) [21].Enhanced cellular uptake has been observed with 100 nm neutral charged (−4 mV) in both cells such as 76% in HUVECs and 56% in HUASMCs compared to the other larger as well as pegylated particles regardless of surface charge.However, negatively charged particles showed the least cell internalization in both cases.To measure the effects of particles with erythrocytes of human blood, percentage of hemolysis was accounted towards different sizes and charges of particles.All types of particles were partially associated with very insignificant consequence on hemolysis (1% or less) without considering either size or surface charge.But, highly anionic charged particles of smaller size resulted insignificant delayed clotting time and platelet activation profile compared to larger particles and other types of charged particles.
Testing blood compatibility of polymeric NPs with human blood is another way for finding the probable adverse effects, which may happen after in vivo administration.To rationalize the hemo-compatibility test, another research group (Mayer et al.) employed PS NPs with variety of sizes and surface charges in different mediums (such as cell culture medium with different FBS ratio, PBS) [29].To assess the influence of polymeric NPs' size and surface charge on human blood, the aim of study was to monitor the adverse effects by measuring complement activation, induction of coagulation, thrombocyte activation, membrane integrity, granulocyte activation, and hemolysis using flow cytometric analysis.Complement (C3a and C5a) levels detection is a consideration of the body's immune system activation.Cationic amidine PS particles were involved with high C3a generation (150.8%).Irrespective to size and surface charge, no NPs were involved with prothrombin level induction.CD62P/CD42b labelling was employed to investigate the thrombocyte activation, which was tested for both low (0.5 mg/mL) and high (2 mg/mL) concentrations.But no thromocytic damage was observed, which were confirmed by no lactate-de-hydrogenase (LDH) release for any of the particles.The percentage of CD11b expression (marker for granulocyte activation) for particle's different sizes and surface charges was reported in that study.Increased percentage of hemolysis for all types of particles was reported using high concentration of particle treatment with human blood.However, larger particles were found less hemolytic than smaller particles, and the most important point was that no influence was observed for negatively charged 160 nm size NPs on erythrocytes of human blood by treating with lower concentration.Overall, positively charged larger particles were involved with more hemolysis compared to negatively charged particles and the latter ones larger than 60 nm size appeared to be less hematotoxic than smaller particles.One interesting finding was; particles resuspended in cell culture medium with 10% fetal bovine serum (FBS) showed less negative zeta potential or about to close to neutral charge compared to the particles resuspended in phosphate buffer saline (PBS).The presence of salts and proteins in the dispersion cell medium might be accountable for neutralizing surface charge of polymeric NPs.
Upon exposure of different types of PLGA NPs to different experimental media, Mura et al. also investigated the possible size and zeta potential variations after resuspension of polymeric NPs in different media with time dependent incubation up to 96 hours at 37˚C [30].Three types of medium such as water, cell culture medium plus 10% FBS and PBS have been considered for evaluation in this experiment.Among different media, water and cell culture medium containing 10% FBS were not involved with significant variations in particle size regardless of surface charge, however after incubation of PLGA/chitosan (CS) NOS in PBS the size was increased.Furthermore, upon exposure to serum containing cell culture medium, PLGA/CS, PLGA/polyvinyl alcohol (PVA), and PLGA/pluronic F-68 (PF-68) NPs did not show any noteworthy change in zeta potential values.
They also designed in vitro model to investigate the toxicity of these prepared three types of NPs with Calu-3 cell line derived from human bronchial adenocarcinoma.This cell line could be a representative bronchial epithelial barrier associated with the discharge of airway mucus substances and the moderation of inflammatory reaction [31] [32].Cell viability responses due to NP treatment with higher concentration after 72 hours incubation demonstrated that only PLGA/PF68 NPs showed progressively decreased cell viability compared to other types of NPs.
From other in vitro studies, it has also been found that NPs with 40 -50 nm size range are involved with maximum uptake [33] [34].However, a recent experiment by Schadlich et al. revealed the effect of size for the accumulation of near-infrared (NIR) fluorescent consisting PLA-PEG polymeric NPs in two tumor xenograft models (HT29 colorectal carcinoma and A2780 ovarian carcinoma) utilizing in vivo fluorescence imaging technique [35].NPs of 111 nm and 141 nm size showed higher biodistribution and accumulation in tumors compared to the larger size (166 nm), which was due to rapid clearance of the larger particles by liver.

Effect of Particle Size and Surface Charge Based on in Vivo Studies
To explore the in vivo effects of specifically sized NPs with respect to surface charge, Kulkarni et al. injected the fluorescent modified and unmodified PS NPs into Sprague-Dawley rats after physicochemical characterization [36].Modification of PS NPs was performed by coating with D-α-tocopheryl polyethylene glycol succinate or Vitamin E TPGS, which was able to switch the zeta potentials of different size NPs to less negative charge.
As previously known, circulating mononuclear phagocytic cells in the bloodstream are the key component of reticuloendothelial system (RES).In addition, RES is also composed of matured cells such as macrophages mainly available in lungs, liver and spleen [37].Studies have shown that the NPs with the size range of 100 to 200 nm could be the optimum range in order to escape the RES recognition [27].Due to rapid clearance from systemic circulation, mostly uncoated NPs were distributed to those organs such as liver and spleen, where mononuclear phagocytic system is located.Consequently, 100 and 200 nm size fluorescent PS-TPGS NPs resulted in higher fluorescence concentration in blood plasma regardless of their surface charges.Liver and spleen were the main target organs, where a substantial decrease in NP distribution was observed for all sizes of TPGS modified PS NPs, since hydrophilic coated surface (stealth effect) possesses the ability to prevent the NPs from RES capture.
Moreover, He et al. in 2010 investigated the effects of size and surface charges on the biodistribution of different sizes of rhodamine B (RhB) labeled carboxymethyl chitosan grafted NPs (RhB-CMCNP) and chitosan hydrochloride grafted NPs (RhB-CHNP) having negative and positive surface charges after intravenous administration into H-22 tumor bearing mice [2].It was clearly demonstrated that biodistribution of 150 nm size NPs having zeta potential of around −15 mV showed higher accumulation at the tumor site and long residence in blood compared to more negative or positive or even larger particles.Due to inflammation and disorder of endothelium along with high demand of nutrient supply, comparatively larger vascular leakage is found in solid tumors, which can provide more access for extravascular targeting macromolecules [38].Low anionic charge (−15 mV) bearing RhB-CMCNP-PS particles exhibited higher percentage of distribution in tumor, which might be due to enhanced residing time during systemic circulation [39].On the other hand, more positive charged NPs could leave the interstitium more competently after arriving the tumor's leaky vasculature leading to be up taken by tumor cells or endothelium adjacent to the endothelium [40].This phenomenon might be the possible reason why high cationic charge (+35 mV) bearing RhB-CHNP particles showed higher percentage of distribution in tumor.Due to enhanced permeability and retention (EPR) effects, smaller particles might be favorable to target the tumor passively due to higher accumulation.However, blood's complement activation system and blood opsonins have been found to fabricate the size of polymeric NPs to a larger extent (>500 nm) resulting rapid blood clearance [39].RhB-CMCNP and RhB-CHNP with larger particle size resulted in higher hepatic disposition.Such higher hepatic disposition could be explained by the investigation performed by Liu et al.They found that NPs with the size range above 300 nm were inclined to be blocked or captured by RES as well as liver sinusoids [41].In addition, NPs with the size range from 200 to 500 (nm) was found mainly unaffected by the splenic physical filtration mechanism [42].This could be attributed to obtain lower percentage of hepatic distribution of RhB-CHNP NPs with the particle size range from 150 to 300 (nm).Besides, no influence was observed in distribution of both RhB-CMCNP and RhB-CHNP particles in kidney owing to their size and surface charge.Due to electrostatic reactivity, positively charge particles had tendency to form aggregates with the cells and proteins present in blood and subsequently the aggregation could be trapped by lung [43].As this experiment revealed, He et al. demonstrated the similar result where more cationic RhB-CHNP NPs distribution was found in lung.NPs of 10 -100 nm size is considered as mainly accepted range to design any NP formulation respective to suitable clearance and biodistribution profile before any in vivo trial [3].However, the upper range of particle size is dependent on the interactions with body's immune systems and the lower range is determined by the limit of kidney filtration.Opsonization of larger particles by responsible proteins (e.g.plasma complement, immunoglobulins) in blood compartment is common to develop hypersensitivity response comparatively against larger foreign particles [44] [45].On the other hand, smaller particles (<5.5 nm) have been found with rapid clearance from the body by kidney's glomerular filtration mechanism [46].
To explore the in vivo effects of different size of NPs, Liu et al. prepared radioisotope labeled liposomes of different sizes (30 -400 nm) to inject into the mice models to observe the biodistribution in blood, liver, spleen, and tumor [47].After four hours of post administration, it was found that about 60% of 100 to 200 nm size particles were found in blood, but only 20% of injected particles with size boundary (>250 nm or <50 nm) were detected in blood.In liver, particle size with 100 nm was associated with 20% accumulation, whereas around 25% distribution in liver was detected for larger particles.In spleen, 40% -50% of the injected dose was detected for larger size (>250 nm) but the percentage of detection was lower for the particle size range below 100 nm.In 2002, Levchenko et al. prepared liposomes of around 200 nm with variety of charged surfaces to evaluate the tissue distribution in mice models [48].The results from this study showed that the negatively charged liposomes with zeta potential of around −40 mV were involved with higher clearance rate from the blood in comparison to liposomes with neutral zeta potential.
In addition, Yamamoto et al. investigated the effect of surface charge of poly (ethylene glycol)-poly (D, L-lactide) block copolymer micelles after injecting into male C57/BL6N mice through the tail vein [49].They prepared the micelles with both neutral (tyrosine) and negative (tyrosineglutamine) functionalities, which did not show any significant variations in blood clearance kinetics.However, the negatively-charged micelles displayed a significant lower distribution in both liver and spleen after four hours of post intravenous injection.Overall effects of NPs size and surface charge could be summarized in Figure 2.

Conclusion
In conclusion, polymeric NPs with size range from 10 to 200 nm might not only escape renal filtration and biliary excretion but also accumulate in tumor utilizing EPR effects.Size range above 200 nm may be related to rapid hepatic clearance and RES recognition.Pegylation strategy could be an ideal option to stealth the polymeric NPs for longer residing time during systemic circulation.After in vivo administration of cationic  polymeric NPs, non-specific interaction may occur with non-specific cells or opsonizing protein in blood compartment due to electrostatic bindings, which may involve unexpected cytotoxicity.In order to reduce such nonspecific surface reactivity or interaction, relatively less negatively charged anionic (almost neutral) polymeric NPs with desired small size might be more rationale than cationic charged particles for a broad spectrum biological aspect.This review will help researchers to correlate the in vitro and in vivo effects of polymeric NPs based on particle size and charge.Further investigation and correlation of other physicochemical parameters could be performed on polymeric NPs to understand their biological effects.

Figure 1 .
Figure 1.Relative sizes of NPs favorable for ingestion through various endocytotic pathways.

Figure 2 .
Figure 2. Relative biocompatibility of polymeric NPs based on the effects of size and surface charge.Abbreviation: MPS (Mononuclear Phagocyte System), RES (Reticuloendothelial System), EPR (Enhanced Permeability and Retention).

Table 1 .
Size and surface charge overview of different polymeric NPs.