Counterion of Chitosan Influences Thermodynamics of Association of siRNA with a Chitosan-Based siRNA Carrier.

PURPOSE: The work aimed to compare quality of a siRNA carrier prepared with chitosan of two different sources having similar degree of deacetylation and molecular weights. Differences were analyzed from thermodynamic characteristics of interactions with siRNA. METHODS: The siRNA carrier (chitosan-coated poly(isobutylcyanoacrylate) nanoparticles) was prepared with home-prepared, CSLab, and commercial, CSCom, chitosans. Chitosan counterion was identified and chitosans CSCommod1 and CSCommod2 were obtained from CSCom exchanging counterion with that found on CSLab. Carrier quality was checked considering the size, zeta potential and siRNA association capacity by gel electrophoresis. Thermodynamic parameters of interactions between siRNA and chitosans in solution or immobilized at the carrier surface were determined by isothermal titration calorimetry (ITC). RESULTS: CSLab and CSCommod2 having a high content of acetate counterion associated better siRNA than CSCom and CSCommod1 which counterion included mainly chloride. ITC measurements indicated that siRNA interactions with chitosan and the siRNA carrier were driven by entropic phenomena including dehydration, but thermodynamic parameters of interactions clearly differed according to the nature of the counterion of chitosan. The influence of chitosan counterions was interpreted considering their different lyotropic character. CONCLUSION: Association of siRNA with our siRNA carrier was influenced by the nature of counterions associated with chitosan. Driven by entropic phenomena including dehydration, interactions were favored by acetate counterion. Although more work would be needed to decipher the influence of the counterion of chitosan during association with siRNA, it was pointed out as a new critical attribute of chitosan to consider while formulating siRNA carrier with this polysaccharide.

Understanding Nanomedicine Size and Biological Response Dependency: What Is the Relevance of Previous Relationships Established on Only Batch-Mode DLS-Measured Sizes?

PURPOSE: Most relationships between size and nanomedicine performance and safety were established before the early 2010s' when batch-mode dynamic light scattering (batch-mode DLS) was the only easy size measurement method for colloids available. They are basis for the rational design of nanomedicines, but misunderstood contrasting results are reported. This work aimed to investigate whether these relationships can be used with confidence knowing that batch-mode DLS can be tricky when measuring sizes of polydisperse systems. METHODS: A polydisperse dispersion of polymer nanoparticles ranging from 100 to 465 nm was synthesized. The particles were separated in 4 fractions by successive centrifugations. The capacity of each fraction and parent dispersion to activate the complement system was evaluated by Crossed immuno-electrophoresis. RESULTS: Each fraction was a population of particles with a distinct size. It showed a different capacity to activate the complement system. Particles of the fractions showing the strongest capacity to activate the complement systems had a different size evaluated by batch-mode DLS then that of the parent particles. CONCLUSION: Particles activating the complement system in the parent dispersion were not those that were detected by batch-mode DLS while measuring its size. This work pointed out that previously established relationships between nanomedicine size and their biological response should be taken with caution if sizes were only measured by batch-mode DLS.

Assessment of Complement Activation by Nanoparticles: Development of a SPR Based Method and Comparison with Current High Throughput Methods.

PURPOSE: A Surface Plasmon Resonance chip (SPR) was developed to study the activation of complement system triggered by nanomaterials in contact with human serum, which is an important concern today to warrant safety of nanomedicines. METHODS: The developed chip was tested for its specificity in complex medium and its longevity of use. It was then employed to assess the release of complement fragments upon incubation of nanoparticles in serum. A comparison was made with other current methods assessing complement activation (µC-IE, ELISA). RESULTS: The SPR chip was found to give a consistent response for C3a release upon activation by nanoparticles. Results were similar to those obtained by µC-IE. However, ELISA detection of iC3b fragments showed an explained high non-specific background. The impact of sample preparation preceding the analysis was assessed with the newly develop SPR method. The removal of nanoparticles before analysis showed an important modification in the obtained response, possibly leading to false negative results. CONCLUSION: The SPR chip developed in this work allows for an automated assessment of complement activation triggered by nanoparticles with possibility of multiplexed analysis. The design of the chip proved to give consistent results of complement activation by nanoparticles.

Serial multiple crossed immunoelectrophoresis at a microscale: A stamp-sized 2D immunoanalysis of protein C3 activation caused by nanoparticles.

Crossed immunoelectrophoresis (C-IE) is used to detect and quantify specific proteins. An application allowed the evaluation of complement system activation by nanomaterials. The work aimed to improve the C-IE toward a higher throughput and less tedious method. A new concept was implemented to prepare and run agarose gels. The first and the second dimension of electrophoresis were performed on a single gel plate, prepared before the beginning of the analysis. Several samples were migrated simultaneously on the same migration line. Up to 35 analyses were run at once, providing stamp-sized electrophoregrams (2.8 × 3 cm(2) ) maintaining the performance of the original method performed on 5 × 7 cm(2) gel slabs. Robustness and precision of the method were demonstrated through a validation approach using ANOVA. Handling, experimental duration, amount of reagents, and overall cost of one analysis were considerably reduced compared to the original method. With the same equipment, seven times more analyses can be performed in one run. C-IE can be used to analyze many types of proteins. The new experimental modalities were suitable for the application developed in the present work that was to evaluate activation of protein C3 of the complement system triggered by nanomaterials.

Multimodal Dispersion of Nanoparticles: A Comprehensive Evaluation of Size Distribution with 9 Size Measurement Methods.

PURPOSE: Evaluation of particle size distribution (PSD) of multimodal dispersion of nanoparticles is a difficult task due to inherent limitations of size measurement methods. The present work reports the evaluation of PSD of a dispersion of poly(isobutylcyanoacrylate) nanoparticles decorated with dextran known as multimodal and developed as nanomedecine. METHODS: The nine methods used were classified as batch particle i.e. Static Light Scattering (SLS) and Dynamic Light Scattering (DLS), single particle i.e. Electron Microscopy (EM), Atomic Force Microscopy (AFM), Tunable Resistive Pulse Sensing (TRPS) and Nanoparticle Tracking Analysis (NTA) and separative particle i.e. Asymmetrical Flow Field-Flow Fractionation coupled with DLS (AsFlFFF) size measurement methods. RESULTS: The multimodal dispersion was identified using AFM, TRPS and NTA and results were consistent with those provided with the method based on a separation step prior to on-line size measurements. None of the light scattering batch methods could reveal the complexity of the PSD of the dispersion. CONCLUSIONS: Difference between PSD obtained from all size measurement methods tested suggested that study of the PSD of multimodal dispersion required to analyze samples by at least one of the single size particle measurement method or a method that uses a separation step prior PSD measurement.

Match of Solubility Parameters Between Oil and Surfactants as a Rational Approach for the Formulation of Microemulsion with a High Dispersed Volume of Copaiba Oil and Low Surfactant Content.

PURPOSE: Aim was to formulate oil-in-water (O/W) microemulsion with a high volume ratio of complex natural oil, i.e. copaiba oil and low surfactant content. The strategy of formulation was based on (i) the selection of surfactants based on predictive calculations of chemical compatibility between their hydrophobic moiety and oil components and (ii) matching the HLB of the surfactants with the required HLB of the oil. METHOD: Solubility parameters of the hydrophobic moiety of the surfactants and of the main components found in the oil were calculated and compared. In turn, required HLB of oils were calculated. Selection of surfactants was achieved matching their solubility parameters with those of oil components. Blends of surfactants were prepared with HLB matching the required HLB of the oils. Oil:water mixtures (15:85 and 25:75) were the titrated with surfactant blends until a microemulsion was formed. RESULTS: Two surfactant blends were identified from the predictive calculation approach. Microemulsions containing up to 19.6% and 13.7% of selected surfactant blends were obtained. CONCLUSION: O/W microemulsions with a high volume fraction of complex natural oil and a reasonable surfactant concentration were formulated. These microemulsions can be proposed as delivery systems for the oral administration of poorly soluble drugs.

Drug-free chitosan coated poly(isobutylcyanoacrylate) nanoparticles are active against trichomonas vaginalis and non-toxic towards pig vaginal mucosa.

PURPOSE: The present work reports a non-conventional therapeutic strategy based on the use of vaginally-applied formulations for the treatment of trichomoniasis due to Trichomonas vaginalis without adding a drug. METHODS: The formulations were based on a thermosensitive pluronic® F127 hydrogel containing mucoadhesive poly(isobutylcyanoacrylate) nanoparticles coated with a mixture of chitosan and thiolated chitosan (75/25 wt%). The nanoparticles were obtained by anionic emulsion polymerization of isobutylcyanoacrylate. The anti-T. vaginalis activity of the formulations was evaluated in vitro. RESULTS: Chitosan-coated nanoparticles showed a strong anti-T. vaginalis activity at 100 µg/mL independently on the proportion of thiolated chitosan. No anti-T. vaginalis activity was reported neither with chitosan-uncoated poly(isobutylcyanoacrylate) nanoparticles nor with chitosan used as a solution. These results suggest that the anti-T. vaginalis activity was related to poly(isobutylcyanoacrylate) nanoparticles but only when they are coated with chitosan. Histological analysis of ex vivo pig vaginal mucosa in contact with pluronic® F127 hydrogel containing poly(isobutylcyanoacrylate) nanoparticles coated with the mixture chitosan/thiolated chitosan (75/25 wt%) did not reveal any toxicity. CONCLUSION: This study demonstrated that poly(isobutylcyanoacrylate) nanoparticles coated with chitosan were active against T. vaginalis without adding a drug. Besides their anti-T. vaginalis activity, the formulations are non-toxic towards pig vaginal mucosa.

Polymer prodrug nanoparticles based on naturally occurring isoprenoid for anticancer therapy.

The synthesis of a novel class of polymer prodrug nanoparticles with anticancer activity is reported by using squalene, a naturally occurring isoprenoid, as a building block by the reversible addition-fragmentation (RAFT) technique. The RAFT agent was functionalized by gemcitabine (Gem) as anticancer drug, and the polymerization of squalenyl-methacrylate (SqMA) led to well-defined macromolecular prodrugs comprising one Gem at the extremity of each polymer chain. The amphiphilic nature of the resulting Gem-PSqMA conjugates allowed them to self-assemble into long-term stable and narrowly dispersed nanoparticles with significant anticancer activity in vitro on various cancer cell lines. To confer stealth properties on these nanoparticles, their PEGylation was successfully performed, as confirmed by X-ray photoelectron spectroscopy (XPS) and complement activation assay. It was also shown that the PEGylated nanoparticles could be internalized in cancer cells to a greater extent than their non-PEGylated counterparts.

The counterbalanced effect of size and surface properties of chitosan-coated poly(isobutylcyanoacrylate) nanoparticles on mucoadhesion due to pluronic F68 addition.

PURPOSE: To evaluate of the effect of size and surface characteristics of poly(isobutylcyanoacrylate) nanoparticles coated with pluronic F68 and thiolated chitosan on mucoadhesion. METHODS: Nanoparticles were obtained by radical emulsion polymerization in presence of different amounts of F68 (0-4%w/v). Mucoadhesion was ex vivo evaluated by applying nanoparticle suspension on rat intestinal mucosa and quantifying the amount of attached nanoparticles after incubation. RESULTS: F68 unimers added in the polymerization medium allowed decreasing nanoparticle size from 251 to 83 nm, but resulted in nanoparticle surface modification. The amount of thiolated chitosan onto nanoparticle surface was decreased resulting in lower thiol groups and zeta potential. Consequently, the decrease of nanoparticle hydrodynamic diameter resulted in eight-fold-increase of the number of nanoparticles attached to the mucosa but a significant decrease of the weight of attached nanoparticles was observed. This unexpected result was due to a decrease of the amount of chitosan and thiolated chitosan available to interact with mucus upon addition of F68 in the polymerization medium. CONCLUSIONS: Addition of F68 should not be recommended to improve the amount of mucoadherent nanoparticles. Further studies could allow understanding if the low amount of small size nanoparticles could be able to improve oral bioavailability.

Nanoparticles facing the gut barrier: Retention or mucosal absorption? Mechanisms and dependency to nanoparticle characteristics.

A better knowledge on influence of nanomedicine characteristics on their biological efficacy and safety is expected to accelerate their clinical translation. This work aimed understanding of the oral fate of polymer-based nanomedicines designed with different characteristics. The influence of nanoparticle characteristics (size, zeta potential, molecular architecture surface design) was explored on biological responses evaluating their retention and absorption by rat jejunum using the Ussing chamber experimental model. Thermodynamic aspects of interactions between nanoparticles and model mucins were elucidated by isothermal titration calorimetry. The retention on mucosa varied between nanoparticles from 18.5 to 97.3 % of the initial amount after a simulation considering the entire jejunum length. Different mechanisms were proposed which promoted mucosal association or oppositely precluded any interactions. Strikingly, mucosal retention was profoundly affected by the size and nature of interactions with the mucus which depended on the nature of the coating material, but not on the zeta potential. The nanoparticle absorption simulated along the whole length of the intestine was low (0.01 to almost 3% of the initial amounts). A saturable mechanism including an upper nanoparticle size limit was evidenced but, needs now to be further elucidated. This work showed that the molecular design and formulation of nanoparticles can guide mechanisms by which nanoparticles interact with the mucosa. The data could be useful to formulators to address different oral drug delivery challenges ranging from the simple increase of residence time and proximity to the absorptive epithelium and systemic delivery using the most absorbed nanoparticles.

New core-shell nanoparticules for the intravenous delivery of siRNA to experimental thyroid papillary carcinoma.

PURPOSE: Development of efficient in vivo delivery nanodevices remains a major challenge to achieve clinical application of siRNA. The present study refers to the conception of core-shell nanoparticles aiming to make possible intravenous administration of chemically unmodified siRNA oriented towards the junction oncogene of the papillary thyroid carcinoma. METHODS: Nanoparticles were prepared by redox radical emulsion polymerization of isobutylcyanoacrylate and isohexylcyanoacrylate with chitosan. The loading of the nanoparticles with siRNA was achieved by adsorption. The biological activity of the siRNA-loaded nanoparticles was assessed on mice bearing a papillary thyroid carcinoma after intratumoral and intravenous administration. RESULTS: Chitosan-coated nanoparticles with a diameter of 60 nm were obtained by adding 3% pluronic in the preparation medium. siRNA were associated with the nanoparticles by surface adsorption. In vivo, the antisense siRNA associated with the nanoparticles lead to a strong antitumoral activity. The tumor growth was almost stopped after intravenous injection of the antisense siRNA-loaded nanoparticles, while in all control experiments, the tumor size was increased by at least 10 times. CONCLUSION: This work showed that poly(alkylcyanoacrylate) nanoparticles coated with chitosan are suitable carriers to achieve in vivo delivery of active siRNA to tumor including after systemic administration.

siRNA nanoformulation against the ret/PTC1 junction oncogene is efficient in an in vivo model of papillary thyroid carcinoma.

Delivery is a very important concern for therapeutic applications of siRNA. In this study, we have used chitosan-coated poly(isobutylcyanoacrylate) nanoparticles to deliver siRNA with a complementary sequence to the fusion oncogene ret/PTC1. By screening the mRNA junction we have selected a potent siRNA sequence able to inhibit this oncogene in a model of Papillary Thyroid Carcinoma cells. This siRNA sequence has then been validated by a shRNA approach using the same sequence. Furthermore, the high ret/PTC1 inhibition has triggered a phenotypic reversion of the transformed cells. We have designed well-defined chitosan decorated nanoparticles and succeeded to reduce their size. They have allowed to protect ret/PTC1 siRNA from in vivo degradation and leading to significant tumour growth inhibition after intratumoral administration.

Elucidation of the complexation mechanism between (+)-usnic acid and cyclodextrins studied by isothermal titration calorimetry and phase-solubility diagram experiments.

In the present work the complexation mechanism between (+)-usnic acid (UA) and cyclodextrins (CDs) has been investigated by isothermal titration calorimetry (ITC) and phase-solubility diagrams using pH as a tool for modifying the molecule ionization. ITC experiments have been employed to evaluate the stoichiometry of interaction (N), affinity constants (K), and thermodynamic parameter variation associated with complexation between (+)-UA and alpha-, beta-, HP-beta-, SBE-beta-, and gamma-CD. It was shown that (+)-UA did not interact with alpha-CD and tended to interact more favorably with gamma-CD (K = 1030 M(-1), DeltaG = -17.18 kJ x mol(-1)) than beta-CD (K = 153 M(-1), DeltaG = -12.46 kJ x mol(-1)) forming 1:1 complexes. It was also demonstrated using ITC and solubilization experiments that chemical modifications of the parent beta-CD resulted in stronger and more spontaneous interactions (K = 281 M(-1), DeltaG = -13.97 kJ x mol(-1) for SBE-beta-CD and K = 405 M(-1), DeltaG = -14.87 kJ x mol(-1) for HP-beta-CD). Analysis of the thermodynamic data suggested that van der Waals forces and hydrogen bonds were responsible for the formation of complexes with a predominance of van der Waals forces. Finally, pH induced modifications of (+)-UA ionization provided important informations relative to the topology of the interaction between (+)-UA molecule and the gamma-CD cavity, which were confirmed by molecular modeling.

PEGylated degradable composite nanoparticles based on mixtures of PEG-b-poly(γ-benzyl L-glutamate) and poly(γ-benzyl L-glutamate).

In the present work, the possibility to obtain PEGylated nanoparticles from two PBLG derivatives, PEG-b-poly(γ-benzyl L-glutamate), PBLG-PEG-60, and poly(γ-benzyl L-glutamate), PBLG-Bnz-50, by nanoprecipitation has been investigated. Particles were prepared not only from one polymer (PBLG-PEG-60 or PBLG-Bnz-50), but also from mixtures of two PBLG derivatives, PBLG-PEG-60 and PBLG-Bnz-50, in different ratios (90/10, 77/23, and 40/60 wt %). Because of the presence of PEG chains, hydrophilic particles were obtained, which was confirmed by ζ potential measurements (ζ from -13 mV and -21 mV) and by isothermal titration microcalorimetry (ITC). This last technique has shown no heat exchange when BSA was added to PEGylated nanoparticles. Further, complement activation has been evaluated by crossed immuno-electrophoresis demonstrating that the introduction of 77 wt % of PEGylated PBLG chains in the particles was enough to ensure a low complement activation activity. This effect was strongly correlated to the ζ potential of the particles, which decreased with an increase of PEG chains content. Interestingly, such properties are of interest for the preparation of degradable stealth nanocarriers. Moreover, it is suggested that the introduction of a reasonable amount (up to 20 wt %) of a second copolymer in the particle composition can be possible without modifying their stealth character. Moreover, the presence of this second copolymer would help to introduce a second functionality to the particles.

Methods for the preparation and manufacture of polymeric nanoparticles.

This review summarizes the different methods of preparation of polymer nanoparticles including nanospheres and nanocapsules. The first part summarizes the basic principle of each method of nanoparticle preparation. It presents the most recent innovations and progresses obtained over the last decade and which were not included in previous reviews on the subject. Strategies for the obtaining of nanoparticles with controlled in vivo fate are described in the second part of the review. A paragraph summarizing scaling up of nanoparticle production and presenting corresponding pilot set-up is considered in the third part of the review. Treatments of nanoparticles, applied after the synthesis, are described in the next part including purification, sterilization, lyophilization and concentration. Finally, methods to obtain labelled nanoparticles for in vitro and in vivo investigations are described in the last part of this review.

A journey through the emergence of nanomedicines with poly(alkylcyanoacrylate) based nanoparticles.

Starting in the late 1970s, the pioneering work of Patrick Couvreur gave birth to the first biodegradable nanoparticles composed of a biodegradable synthetic polymer. These nanoparticles, made of poly(alkylcyanoacrylate) (PACA), were the first synthetic polymer-based nanoparticulate drug carriers undergoing a phase III clinical trial so far. Analyzing the journey from the birth of PACA nanoparticles to their clinical evaluation, this paper highlights their remarkable adaptability to bypass various drug delivery challenges found on the way. At present, PACA nanoparticles include a wide range of nanoparticles that can associate drugs of different chemical nature and can be administered in vivo by different routes. The most recent technologies giving the nanoparticles customised functions could also be implemented on this family of nanoparticles. Through different examples, this paper discusses the seminal role of the PACA nanoparticles' family in the development of nanomedicines.

Preparation of a Carrier to Achieve In Vivo Delivery of siRNA: The Example of Chitosan-Coated Poly(isobutylcyanoacrylate) Nanoparticles.

This chapter describes the preparation of chitosan-coated poly(isobutylcyanoacrylate) nanoparticles as a suitable carrier to deliver siRNAs to two types of xenograft tumor models of mice. The nanoparticles are prepared by a method of emulsion polymerization that includes steps of polymerization and purification. The polymerization method is carried out in a single pot in an aqueous medium. siRNAs are coupled with the nanoparticles at the end of the preparation by adsorption. The protocol also explains how to determine optimum yield/the titer of association of siRNA with the nanoparticles. It is described for a preparation scale at 4 mL of nanoparticle dispersion at a concentration of 42-46 mg nanoparticles/mL. Optimal loading capacity of the nanoparticles with the siRNA can be achieved by performing an association yield above 90% using a mass ratio of 1 mg siRNA/50 mg of nanoparticles (20 µg siRNA/mg nanoparticles, 1 nmol siRNA (Mw 14 kDa)/mg nanoparticles).

Characterization of nanomedicines: A reflection on a field under construction needed for clinical translation success.

The nanotechnology revolution offers many expectations for the improvement of medicine treatments. At present, nanomedicine (NM) development is hampered by methodological barriers for a better characterization and a wider understanding of their in vivo behavior. While regulatory agencies setup guidelines to support NM translation from bench to bedside, the gap is still hardly overcome by main nanomedicines. One lever for filling this gap is a better characterization, thus increasing the global knowledge about the NM itself but also validate the confidence in terms of batch to batch reproducibility of such complex nano-objects. Here, we review the current methodologies routinely used for clinical release of nanomedicine batches in compliance with official guidelines. We confront them to the extreme sharpness of biological systems and finally discuss future possible orientations for a better characterization of NMs, needed to bridge the gap between physicochemical properties and biological fate.

Mucoadhesion mechanism of chitosan and thiolated chitosan-poly(isobutyl cyanoacrylate) core-shell nanoparticles.

The study is focused on the evaluation of the potential bioadhesive behaviour of chitosan and thiolated chitosan (chitosan-TBA)-coated poly(isobutyl cyanoacrylates) (PIBCA) nanoparticles. Nanoparticles were obtained by radical emulsion polymerisation with chitosan of different molecular weight and with different proportions of chitosan/chitosan-TBA. Mucoadhesion was ex vivo evaluated under static conditions by applying nanoparticle suspensions on rat intestinal mucosal surfaces and evaluating the amount of nanoparticles remaining attached to the mucosa after incubation. The analysis of the results obtained demonstrated that the presence of either chitosan or thiolated chitosan on the PIBCA nanoparticle surface clearly enhanced the mucoadhesion behaviour thanks to non-covalent interactions (ionic interaction and hydrogen bonds) with mucus chains. Both, the molecular weight of chitosan and the proportion of chitosan-TBA in the formulation influenced the nanoparticle hydrodynamic diameter and hence their transport through the mucus layer. Improved interpenetration ability with the mucus chain during the attachment process was suggested for the chitosan of high molecular weight, enhancing the bioadhesiveness of the system. The presence of thiol groups on the nanoparticle surface at high concentration (200 x 10(-6) micromol SH/cm2) increased the mucoadhesion capacity of nanoparticles by forming covalent bonds with the cysteine residues of the mucus glycoproteins.

PEGylation of microspheres for therapeutic embolization: preparation, characterization and biological performance evaluation.

In this study, microspheres designed for embolization, defined as GF2000-Trisacryl MS (GF-MS) and DEAE-Trisacryl MS (DEAE-MS), were originally PEGylated using (3-amino propyl) triethoxy silane as coupling agent. Indomethacin was loaded into both PEGylated and non-PEGylated DEAE-MS, displaying ion-exchange ability, through a batch process with a respective capacity of 1.2 and 0.25 g/g. The morphology of naked and PEGylated MS was evaluated by scanning electron microscopy (SEM). Both micosphere resins surface looked like orange skin, although DEAE-MS showed a slightly rougher surface due to the copolymerization process. PEGylated microspheres have a most likely swelling surface owing to the presence of PEG hydrophilic chains. The mean diameters were of about 66 and 60 microm for GF-MS and DEAE-MS, respectively. Data obtained for PEGylated MS by Fourier Transform Infrared spectroscopy (FTIR) confirmed that microspheres were successfully PEGylated. Finally, complement activation in vitro was performed to evaluate the activating capacity of different microspheres. Both PEGylated GF-MS and DEAE-MS activated the complement system of about 33% less than their corresponding naked microspheres, while loading PEGylated DEAE-MS with indomethacin almost suppressed complement activation. This inhibiting role implies that PEGylation as well as loading the microspheres with anti-inflammatory drug has a compact effect on the interaction of microspheres with blood proteins.