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Current Nanoscience, 2016, 12, 347-356
Smart Biodegradable Nanoparticulate Materials: Poly-lactide-co-glycolide Functionalization with Selected Peptides
ISSN: 1573-4137 eISSN: 1875-6786
Barbara Colzani1, Marco Biagiotti2, Giovanna Speranza2, Rossella Dorati1, Tiziana Modena1, Bice Conti1, Corrado Tomasi3 and Ida Genta*,1 1
Department of Drug Sciences, University of Pavia, Pavia, Viale Taramelli, 27100, Pavia – Italy; Dept. Chemistry, University of Milan, 19, Via Golgi, 2013, Milano – Italy; 3I.E.N.I. C.N.R., Unit of Lecco, 29, C.so Promessi Sposi, 23900, Lecco, Italy
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Abstract: Background: Smart nanoparticulate materials, namely tailored nanoparticles (NPs) with specific surface functionality, have recently attracted attention as useful tool for time- and site-specific drug delivery. Specifically, polymeric nanoparticles (NPs) can be chemically functionalized with different chemical entities, i.e. peptides, that selectively recognize biological substrates in vivo and target drug release. Divergent and very complex strategies can be pursued in order to obtain peptidedecorated NPs.
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Methods: A simple method was suggested for direct functionalization of poly-lactide-co-glicolide (PLGA) with small peptides. A solid-phase peptide synthesis was used to obtain a dodecapeptide (GE11) and a smaller tetrapeptide (FQPV). FQPV- and GE11-PLGA conjugates were obtained by optimized carbodiimide chemistry. Nanoprecipitation and solvent extraction/evaporation methods were purpose-built in order to prepare FQPV-PLGA NPs. NPs were characterized in terms of size, surface charge and adsorbed peptide amount; ex-vivo cytotoxicity studies were performed on FQPV-PLA NPs using adult fibroblasts.
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Results: Custom GE11, recently known as efficient Epidermal Growth Factor Receptor targeting agent, and FQPV, used as model peptide, were synthesized by solid-phase peptide synthesis achieving good purity (95%) and satisfactory process yields (70-85%). Then, FQPV- and GE11-PLGA conjugates were obtained by optimized carbodiimide chemistry achieving an high degree of functionalization (> 85%). Aware of different physico-chemical properties of peptide-PLGA conjugates with respect to plain PLGA, two different NPs preparation techniques, nanoprecipitation and solvent extraction/evaporation methods, were purpose-built in order to prepare FQPV-PLGA NPs. Both methods revealed suitable to obtain NPs with proper dimensions for the parenteral administration (< 250nm), narrow size distribution (P.I. about 0.1), good morphological features and negative charge (about –20mV). A peptide adsorption protocol onto NPs was considered as additional strategy to increase peptide expression on NPs surface aimed at improving the targeting effectiveness. A Design of Experiment approach (DoE) has been successfully applied to the more versatile solvent extraction/evaporation method in order to systematically highlight the influence of process parameters (organic solvent mixture, PVA concentration and polymeric solution volume) on NPs sizes.
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Conclusion: PLGA was successfully functionalized with two different peptides, FQPV and GE11, following the a versatile and simple carbodiimmide chemistry without further modifying polymer and/or peptide structure. Both nanoprecipitation and solvent extraction/evaporation NPs preparation methods were properly optimized in order to obtain peptide-PLGA based NPs and they can be alternatively selected according to solubility properties of both peptide-polymer conjugate and drug intended for encapsulation. Peptide adsorption on preformed PLGA NPs could be efficiently used to increase peptide expression on NPs surface thus improving cellular recognition in case of active targeting. Preliminary cytocompatibility evaluation of the selected peptide-PLGA based nanoparticulate materials shows a potential feasibility of the set-up synthetic procedures and NPs preparation methods for pharmaceutical purposes.
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Keywords: FQPV-PLGA nanoparticles, GE11-PLGA nanoparticles, peptide synthesis, peptide-PLGA conjugates, peptidepolymer conjugate synthesis, PLGA nanoparticles, smart PLGA nanomaterials. 1. INTRODUCTION Nanotechnology is generally defined as the engineering and manufacturing of materials at the atomic and molecular scale [1]. In the pharmaceutical field the application of *Address correspondence to this author at the Department of Drug Sciences, University of Pavia, Viale Taramelli, 12, 27100 Pavia, Italy; Tel/Fax: +39 0382 987371; E-mail:
[email protected] 1875-6786/16 $58.00+.00
nanotechnology focuses on drug delivery and aims to alter the characteristics of a therapeutic molecule to increase solubility, decrease degradation during circulation, deliver to active site at the right time in a therapeutically effective concentration promising to increase efficacy while decreasing unwanted side effects [2]. In the nanoscaled drug delivery devices field the ability to apply polymeric nanomaterials as delivery agents for drugs and other therapeutics are at the © 2016 Bentham Science Publishers
Impact Factor: 1.096
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forefront of projects in nanomedicine and are beginning to expand the market for many drugs to make a significant impact on global pharmaceutical planning and marketing [3, 4] holding promise for a wide variety of diseases, including many types of cancer [5], cardiovascular diseases [6], diabetes [7, 8], some chronic metabolic syndromes and degenerative diseases and disorders [9].
only few studies have been reported on GE11-functionalized biodegradable nanoparticulate materials and in which GE11polymer conjugate was obtained by time-consuming and multistep synthesis procedures [20, 21]. In the present study, polylactide-co-glicolide (PLGA) is chosen as polymeric backbone; PLGAs represent a versatile family of well-recognized biocompatible and biodegradable polymers and FDA authorized for parenteral administration in humans [22]. Custom GE11 and a smaller tetrapeptide (FQPV), used as model peptide, were synthesized by a solid phase peptide synthesis method and FQPV- and GE11PLGA conjugates were obtained by optimized carbodiimide chemistry in order to set-up a simple and efficient method to directly conjugate PLGA to peptides by reaction of the carboxylic end group of the polymer with the N-terminal amino groups of the peptides. Due to variations of the peptide-PLGA physico-chemical properties with respect to plain polymer, the choice and optimization of peptide-PLGA NPs preparation technique can be challenging. Two different and extensively used PLGA NPs preparation techniques, nanoprecipitation and solvent extraction/evaporation methods [23, 24], were purpose-built for FQPV-PLGA conjugate; furthermore, thanks to higher versatility of the solvent extraction/ evaporation method with respect to NPs encapsulation efficiency of potential therapeutic agents with different solubility properties, this technique was systematically validated by a design of experiment (DoE) approach.
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The rapid advancement in the above direction has been made with the development of advanced nanofabrication techniques to produce conjugates of peptide/proteins to polymers, the most prominent representatives of smart nanomaterials both in therapeutic and diagnostic area [1517]. Divergent can be the approaches for preparing this class of conjugates including peptide synthesis from synthetic polymer supports, polymerization from peptide/protein macroinitiators or chain transfer agents and the polymerization of peptide side-chain monomers but simplicity needs to be the paradigm. It has been shown that the development of targeted drug delivery nanosystems by self-assembly of prefunctionalized biomaterials simplifies the optimization and the potential manufacturing of these advanced nanomaterials.
The rationale is to obtain a biodegradable, biocompatible smart nanoplatforms using well-characterized and safe raw materials by simple but efficient and versatile approaches to both peptide-polymer conjugate synthesis and NPs preparation method.
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More recently smart nanoparticulate systems, namely tailored nanoparticles (NPs) with specific surface functionality, have attracted attention due to their unique properties and potential usefulness when compared to conventional particulate systems [12, 13]. Well-suited for parenteral administration, smart nanoparticulate materials consist of multi-functionalized polymeric carriers in which a biocompatible, non-immunogenic, biodegradable polymer has been converting in an advanced bioresponsive polymer thanks to a conjugation with a targeting mojety (i.e. peptides, proteins, antibody fragments etc). This entity is able to selectively address the nanostructures to their biological target, permitting site-specific drug delivery modulated by the polymer itself or triggered by other interactive components of the engineered nanomaterials (stimuli responsive smart nanoparticulate systems) [14].
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It is becoming increasingly evident that optimal nanoscale drug delivery systems must have certain critical morphological, physicochemical and biological attributes [10, 11]; simultaneously, it should be versatile nanoparticulate platforms providing delivery for many different drugs, made of materials that can meet the Regulatory Authority requirements and prepared by simple and easily scalable manufacturing process [12].
In this perspective this work aims to set-up simple chemical and technological protocols to obtain a biodegradable peptide-polymer conjugate based nanoparticulate platform as smart material potentially intended for selective targeting to cells overexpressing Epidermal Growth Factor Receptor (EGF-R). EGF-R overexpression represents a peculiar feature in diseases characterized by fast growing tissue, like cancers [18]; this receptor is quite well characterized in structural terms permitting to identify novel and selective EGF-R peptide ligands which bind to the receptor, internalize but does not activate the EGF-R. Among them the dodecapeptide YHWYGYTPQNVI (GE11) has been identified as one of the more effective EGF-R targeting agents [19]. To date,
2. EXPERIMENTAL 2.1. Materials PLGA (poly-(D,L-lactide-co-glicolide), 75:25, MW 30 KDa) was purchased from Lakeshore Biomaterials. All other reagents, resins and solvents were purchased from Sigma-Aldrich (Saint Louis, MO, USA) and/or from Merck KGaA (Darmstadt, Germany) were used without further purification; all the solvents were of HPLC grade. 2.2. Synthesis of Peptides Before the synthesis of GE11, a small tetrapeptide (FQPV) was selected as model peptide to set up the best synthetic protocol. FQPV peptide (Fig. 1a) was obtained by Microwave assisted solid phase automated synthesis using fluorenylmethyloxycarbonyl (Fmoc)-protocol on a Biotage SP Wave Initiator+ synthesizer (Biotage, Uppsale, Sweden) and a preloaded Fmoc-Val-2-Cl-Trityl resin, swelled with dimethylformamide (DMF). Synthesis was carried out on 0.2 mmol scale (450 mg of loaded resin). Each Fmoc deprotection was performed using a 25% pyperidine in DMF solution.
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Each coupling reaction was carried out at 50°C in DMF using hydroxybenzotriazole (HOBt) (41 mg, 0.60 mmol) and N,N,N’,N’-Tetramethyl-O-(1H-benzotriazol-1-yl)uronium hexafluorophosphate (HBTU) (114 mg, 0.60 mmol) as coupling agents and N,N-Diisopropylethylamine (DIPEA) (0.10 mL, 1.20 mmol) as base; reaction time was 15 minutes. Amino acids (AA) were used in a 3 fold excess (0.60 mmol): in particular 203 mg of Fmoc-Pro-OH, 367 mg of FmocGln(Trt)-OH, 233 mg of Fmoc-Phe-OH were used.
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mass spectrometry (ESI-MS) were used to check the identity of the peptides. MALDI-TOF spectra were acquired on a Bruker Microflex LT Spectrometer Electrospray ionization mass (Bruker, Billerica, MA, USA) and ESI-MS spectra were recorded on a Thermo Finnigan LCQ Advantage spectrometer (Hemel Hempstead, Hertfordshire, UK). 2.4. Preparation of the Polymer-peptide Conjugates
Each amino acid was dissolved, along with coupling agents and DIPEA, in DMF (3 mL) 15 minutes before the reaction.
2.3. Peptide Purification and Characterization
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2.5. Characterization of Polymer-peptide Conjugates
The 1H NMR spectra of peptide-polymer conjugates (FQPV- and GE11-PLGA) were acquired at 400.13 MHz in DMSO-d6 at 50°C on a Bruker Advance 400 spectrometer (Bruker, Karlsruhe, Germany) interfaced with a workstation running Windows operating systems and equipped with a TopSpin software package. Chemical shifts are given in ppm () and are referenced to DMSO signal as internal standard (H DMSO 2.5 ppm). Through the comparison of peak integration area value in the NMR spectrum, it was possible to establish PLGA functionalization with peptides.
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Peptides purification was performed by HPLC, using an Amersham Pharmacia Biotech (P900) liquid chromatographer connected to a UV-vis detector (GE Healthcare, Little Chalfont, Buckinghamshire, UK); chromatographic conditions were set as follows: column for analytical HPLC, Jupiter RP-18 (10m proteo 90A size: 250x4.60 mm, Phenomenex, Torrance, CA); column for semipreparative HPLC, Jupiter- RP-18 (10 m, size:250x10 mm, Phenomenex, Torrance, CA); detector, 226 and 254 nm; mobile phase: A (0.1% TFA (v/v) in water) and B (80% acetonitrile/ 20% water with 0.1% of TFA), gradient elution from 5% to 40% B in 3 column volumes (cv), from 40% to 70% B in 3 cv, from 70% to 100% B in cv and finally 2cv at 100% B.
The same procedure was also used to functionalize the activated PLGA with GE11, thus obtaining GE11-PLGA conjugate.
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GE11 peptide (YHWYGYTPQNVI, Fig 1b) was synthesized following the same protocol as described previously: a Microwave assisted solid phase automated synthesis was performed on Biotage SP Wave Initiator+ synthetsizer (Biotage, Uppsale, Sweden) using a Fmocprotected amino acids and preloaded Fmoc-Ile-2-Cl-Trityl resin.
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For cleavage a trifluoroacetic acid (TFA) (17,60 ml)/ phenol (1.00 g)/H2O (1.00 ml)/ triisopropylsilane (TIPS) (0,40 ml) solution was used: the resin was transferred in a round bottom flask and treated with the cleavage solution for about 1 hour. The solid support was then removed by filtration and the resulting oil was diluted with ethyl ether at 0°C. The solvent was removed in vacuo and the residue treated with 1:1 hexane:ethyl ether mixture and left overnight at -15°C. Raw product was collected as white solid and purified by HPLC.
PLGA (50 mg, 1.6710-3 mmol) was dissolved in anhydrous dichloromethane (DCM) (2 mL) under nitrogen atmosphere. DIPEA (0.03 mL, 1.6710-1 mmol), 1-Ethyl3-(3-dimethylaminopropyl)carbodiimide (EDCHCl) (16.2 mg, 8.3510-2 mmol) and, after 15 minutes, Nhydroxysuccinimide (NHS, 9.6 mg, 8.3510-2 mmol) were added. The reaction mixture was left at r.t. under magnetic stirring for 24 hours and then washed with water and brine. Organic phase was anhydrified using sodium sulphate and dried under reduced pressure. Activated PLGA (50 mg, 2.2010-3 mmol) was dissolved in acetonitrile (1.5 mL). To a solution of FQPV peptide (11 mg, 2210-3 mmol), in acetonitrile (1.5 mL) containing few drops of DIPEA, the activated PLGA solution was added and the mixture was left at r.t. under magnetic stirring for 24 hours. The acetonitrile was removed and the residue was dissolved in DCM (5 mL) and washed with a NaOH solution (0.5 M, 2 times with 3 mL each), water (3 mL) and brine (3 mL). The organic phase was anhydrified using sodium sulphate and dried under reduced pressure recovering the FQPV-PLGA conjugate.
Both Matrix assisted laser desorption/ionization spectrometry (MALDI-TOF) and electrospray ionization
Fig. (1). Primary structure of the two peptides a) FQPV and b) GE11.
The results were expressed in terms of functionalization degree percentage (FD%) as follows: FD% = estimated linkedpeptide x100 theoretical linkedpeptide
(1)
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where “estimated linked-peptide” is the effective amount of peptide linked to PLGA as deduced from NMR spectra and “theoretical linked-peptide” refers to the calculated peptide amount in the peptide-PLGA conjugate considering the mean molecular weights of peptide and PLGA and their 1:1 molar ratio in the conjugate; and weight ratio (WR) expressed as:
The results of the experimental design were analyzed with a suitable software (Statgraphics Centurion XVI.I). 2.7. Recovery of FQPV-PLGA Nanoparticles For both preparation methods NPs were recovered by high speed ultracentrifugation at 30,000 rpm for 30 min at 4°C (Ultracentrifuge Beckman LE80 equipped with a fixed angle rotor Ti70, Beckman Coulter, Pasadena, CA, USA). Each batch was resuspended in bi-distilled water, frozen at 25°C overnight and freeze dried at -50°C, 0.01 bar for 24 h (Lio 5P, Cinquepascal srl, Italy).
estimated linkedpeptide (2) peptidePLGA conjugate where “peptide-PLGA conjugate” is the recovered conjugate weight. WR =
Differential Scanning Calorimetry (DSC) analyses were performed on FQPV, naïve PLGA and FQPV-PLGA conjugate by means of a Modulated Differential Scanning Calorimeter, MDSC 2910 (TA instrument, New Castle, DE, USA). For DSC analysis, 5 mg samples were put into standard aluminium pans and heated from -100°C to 200°C, with an heating rate of 5°C/min, under constant purging of N2 at 20 ml/min. A closed empty pan was used as a reference. The data were treated with Thermal Solutions software (TA Instruments, New Castle, DE, USA) and the results expressed as the mean of three determination.
The process yields of NPs preparation methods were gravimetrically determined and calculated as: Process yield (%) = (NPs /Peptide-PLGA) x100
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where “NPs” is the weight of the NPs batch (after freezedrying) and “peptide-PLGA” is the weight of the employed peptide-PLGA conjugate.
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2.8. Characterization of FQPV-PLGA Nanoparticles
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NPs were submitted to DSC analysis, at the same conditions as described before for peptide-PLGA conjugates (see 2.5 Section).
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According to the first optimized procedure, FQPV-PLGA conjugate was dissolved in DMSO, at a concentration of 15 mg/ml. 5 ml of the polymeric solution were dropped at constant rate (1 ml/min) in 10 ml of 1% w/v PVA aqueous solution under magnetic stirring (700 rpm) for 4 hours. Solid NPs precipitated rapidly, due to the fast diffusion of DMSO in the aqueous phase.
Each NPs batch was submitted to a dimensional analysis in terms of size and polydispersity index (PI) based on dynamic light scattering (DLS) using NICOMP 380 ZLS apparatus (Particle Sizing Systems, CA, USA). Measurements were performed on NPs in a diluted aqueous suspension. The same instrument was also used to evaluate surface charge of NPs suspended in 10 mM NaCl aqueous solution.
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Polymeric NPs based on FQPV-PLGA conjugate were obtained with two different techniques: nanoprecipitation and solvent extraction/evaporation methods [23, 24].
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2.6. Preparation of FQPV-PLGA Nanoparticles
Morphologic characterization of NPs was performed by Transmission Electron Microscopy (TEM 208 S, Philips Eindhoven, NL).
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Solvent extraction/evaporation method consisted in dissolving FQPV-PLGA (15 mg/ml) in a mixture of two different organic solvents, DCM and Acetone, at different composition (v:v). Different volumes of polymeric solution were subsequently emulsified in 10 ml of PVA aqueous solution, at different concentrations, under gentle magnetic stirring (700 rpm) at r.t. for 4 hours. Solvent extraction/ evaporation method was set up by an experimental design approach (see 2.6.1 Section). The rapid diffusion of acetone in the aqueous phase resulted in fast deposition of polymeric film on the droplets and the prolonged stirring time at room temperature permitted to evaporate the organic solvents. With both methods plain PLGA NPs were prepared as reference. All batches were prepared in triplicate. 2.6.1. Experimental Design Approach (DoE) For the solvent extraction/evaporation preparation method a 23 full factorial experimental design was planned and performed thus evaluating the effect of the combination of different independent parameters on NPs sizes (Y), such as composition of DCM:Acetone mixture (X1), concentration of the PVA aqueous solution (X2) and volume of the polymeric solution (X3). The concentration of the polymer was maintained at 15 mg/ml and the volume of the PVA solution was kept at 10 ml.
2.9. FQPV Adsorption on PLGA Nanoparticles Equal amounts of PLGA NPs (3 mg), prepared by nanoprecipitation method, were incubated for different time intervals (10, 30, 60, 90, 120, 180, 240 minutes) with 2 ml of an aqueous solution of FQPV, 2 mg/ml. At each time point, NPs were centrifuged (30,000 rpm, 30 min, 4°C Ultracentrifuge Beckman LE80, fixed angle rotor Ti70) and the supernatants were analyzed to quantify FQPV in solution by an appropriately modified HPLC method [26]. Briefly, the analytical method consists in a reverse-phase high-performance liquid chromatography (Agilent 1260 Infinity, Agilent Technologies, Santa Clara, Ca, USA), with a Zorbax Eclipse Plus C-18 column (4.6x150 mm, 5 m, Agilent). A linear gradient elution using 0.1% v/v TFA in water (A) and 0.1% v/v TFA in Acetonitrile (B) was performed, running from 80:20 to 70:30 (A:B, v/v), at a flow rate of 1 ml/min. FQPV was eluted after 6 minutes; the total run time was 16 minutes. Chromatograms were detected at 215 nm with a multiwave detector Agilent 1260 Infinity (Agilent Technologiesc, Santa Clara, Ca, USA). A good linearity was obtained in the range of 125 and 1500 g/ml (R2=0.997).
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The amount of adsorbed FQPV was calculated as difference between the amount of FQPV in solution (4 mg) and of remaining FQPV in the supernatants after incubation with PLGA NPs.
DMF washing step was performed. After last coupling, resin was washed with DCM and the peptide was finally cleaved from resin support using a mixture of TFA and scavenging agents (88% TFA, 5% PhOH, 5% water and 2% TIPS).
The degree of peptide adsorption (DPA, w/w) was expressed as ratio between the calculated adsorbed FQPV and the recovered PLGA NPs, at each time point:
The raw products were purified by semi-preparative HPLC, using 0.1% v/v TFA in water as the primary eluent and performing a gradient of a 80:20 acetonitrile/0.1% TFA in water solution. The final products were analyzed by MALDI-TOF and/or ESI mass spectrometry: as an example, Fig. (2) shows ESI-MS and MALDI-TOF spectra respectively for FQPV (Fig. 2a) and GE11 (Fig. 2b).
DPA = adsorbed FQPV / PLGA NPs
(4)
2.10. Ex-vivo NP Cytotoxicity The effects of FQPV-PLGA NPs, prepared by both nanoprecipitation and solvent extraction/evaporation method, and PLGA NPs, presenting FQPV absorbed onto the surface (60 min incubation time, see 2.9 Section), on cell viability were assessed with MTT assay [25], using 96 Well Cell Culture Cluster with 10,000 fibroblasts plated in contact to different amounts of NPs corresponding to PLGA concentrations ranging from 0.016 to 10.652 mg/ml. The results were read on a multiwell scanning spectrophotometer (microplate Reader Model 680, Bio-Rad Laboratories, USA). The absorbance was measured at 595 nm with 655 nm as reference wavelength. Cell viability was calculated as percentage of untreated cells (control).
FQPV and GE11 purity was verified using analytical HPLC in the same condition already described for the purification. For both peptides, the purity was more than 95%.
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3. RESULTS AND DISCUSSION
Comment 1 Comment 2
90
Intens. [a.u]
N
95
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14-12#13-42 RT: 0.47-0.76 AV: 9 NL: 4.16E6 ESI Full ms [50.00-2000.00] -490.4
The first step of the synthesis of the FQPV-polymer conjugate consisted in the activation of PLGA using EDC/NHS in DCM (process yield: 84%); then, we isolated the active ester and performed the coupling reaction with
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Both peptides, FQPV and GE11 (Fig. 1a and b), were prepared by solid-phase peptide synthesis method using 2chlorotrytil chloride resin as solid support; the first AA was loaded on the resin using DIPEA in anhydrous DCM [27]. All the synthesis were performed using a semiautomated microwave peptide synthesizer; each Fmoc deprotection was carried out at r.t. treating twice the resin with an adequate amount of a 25% piperidine in DMF solution for 5 and 15 minutes respectively, and then washing the resin with DMF. 15 minutes before each coupling reaction, each AA was dissolved in DMF, along with a 3 fold excess of coupling agents (HOBt and HBTU) and a 6 fold excess of DIPEA as base. The coupling reaction was then carried out under MW irradiation (50 °C) for 20 minutes. After each coupling, a 100
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FQPV was selected as model peptide to optimize the best synthetic procedure to functionalize the polymer with peptides acting as targeting agents, like GE11. For the coupling reaction of the FQPV-PLGA we followed an amide bond formation strategy based on the use of EDC and NHS as schematically reported in Fig. (3). This method is usually preferred over other alternatives because it is quite simple and it takes advantage only of the native functional groups of both PLGA (carboxylic end group) and peptides (N-terminal amino groups) to achieve the coupling, thus resulting in a lower probability to loose ligand specific activity [28]. However, the presence of multiple functional groups in the ligand can result in the possibility of multi-site attachment, thus making difficult the control of the ligand orientation at the surface of the nanocarrier. In our case, FQPV and GE11 peptides bring only one group, the N-terminal primary amine, prone to the formation of an amide bond with PLGA via carbodiimide activation. Therefore, this method appear to be extremely suitable to the formation of the desired conjugate.
85 80
GE11-TA-HCCA(TA)-29-01-2014\0_D2\1\1SLin
1541.2
6000 5000 4000
75 3000
70
2000
60 1000
55
995.6
1001.5
50
Intens. [a.u]
Relative Abundance
65
1045.6
45 40 35 491.4 512.5
30
979.5
25
6000
15
5 0
a
4000
1175.3 1055.6
513.6 186.1
215.3 200
276.1 359.2 489.3 400
535.5 670.2
806.1 789.9 807.3 977.4 800
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GE11-TA-SIN(TA)-29-01-2014\0_D2\1\1SLin
1542.3 8000
1046.7
20
10
0
1058.6 1071.6
1000 m/z
1176.6 1550.8 1557.7 1686.5
1193.5 1311.4 1197.4 1200
1454.3 1536.6 1400
1600
2000 1823.5 1927.5 1800
0
b
500
1000
2122.8
1748.5
1378.9
806.5
2000
1500
2000
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Fig. (2). Mass spectra of a) FQPV and b) GE11 acquired respectively with ESI-MS and MALDI-TOF. O
O O
HO O
OH NHS, EDC, DIPEA, overnight
O
HO
Fig. (3). Scheme of FQPV-PLGA conjugate preparation steps.
O
O O N O
O FQPV, DIPEA, overnight
O
HO O
NHFQPV
3000
3500
m/z
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FQPV peptide dissolved in a mixture of water and acetonitrile. The FQPV-PLGA conjugate was obtained with a process yield of 86%. The isolation of the activated polymer and the change of solvent, although not convenient in term of synthetic efficiency, were necessary to conciliate the different solubility of PLGA (soluble in organic solvents but insoluble in water) and peptide (soluble in water and barely soluble in only few organic solvents).
prepare GE11-PLGA NPs for active targeting through a parenteral administration. Firstly, FQPV-PLGA NPs were prepared according to the nanoprecipitation method. Polymer was dissolved into an organic solvent miscible with water; the polymeric solution was then dropped into an external aqueous solution with a surfactant, under vigorously stirring: the rapid diffusion of the solvent allowed the rapid formation of NPs. Acetone is the solvent commonly used for this procedure, but FQPV-PLGA conjugate demonstrated to be insoluble in this solvent. DMSO was selected as a good alternative since it is water miscible and it is a class III solvent for ICH Q3C “Impurities: guideline for residual solvents”. Briefly, NPs obtained with a FQPV-PLGA concentration of 15 mg/ml in DMSO and a 1% w/v PVA aqueous solution, as external phase, demonstrated suitable sizes, around 202 nm, and a good size distribution as demonstrated by the low PI value (0.133) as shown in Table 1 (# 2- FQPV-PLGA).
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We used 1H NMR spectroscopy to confirm the formation of the peptide polymer bond (see for example Fig. 4) and to give a rough estimation of the peptide-functionalization of the recovered polymer. To this last purpose, we compared the integration area value of the peak at about 0.9 ppm assignable to the peptide with that of the peak at about 5.3 ppm that can be ascribed to the polymer. Knowing both the lactic acid/glycolic acid ratio (75/25) and the average Mw (30 kDa) of the polymer, it was possible to calibrate integrals and calculate the FD% of about 95%, equivalent to a 1:61.47 WR calculated as in Eq. 2.
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This method was optimized following an experimental design approach (DoE) with the aim of evaluating the effect of different parameters and their combinations on the dimensions of NPs. The experimental design was a full factorial screening design with 3 independent factors (X1: composition of DCM:Acetone mixture, X2 : PVA solution concentration and X3: volume of the polymeric solution) and 1 response (Y: NPs size). The other parameters (volume of PVA solution and polymeric solution concentration) were kept constant. Ten NPs batches (23 full factorial and 2 central points) were realized changing the combination of the three
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Fig. (4). a) 1H NMR spectrum of FQPV-PLGA conjugate; b) 1 H NMR spectrum of PLGA. Both spectra were recorded at 400 MHz in DMSO–d6 at 50°C.
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The same protocols were used to realize GE11-PLGA conjugate. In this case, process yield was about 60-70 % w/w and the FD% about 85% w/w. FQPV-PLGA was exploited to set up suitable preparation procedures to obtain NPs with the proper dimensions (< 250 nm). The selected strategies could then be adopted to Table 1.
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For this procedure, a mixture of two solvents was selected, acetone and DCM, characterized by a different water miscibility. Therefore, NPs formation is both due to the diffusion/extraction of acetone in water and to the evaporation of DCM.
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An alternative method, namely solvent extraction/ evaporation method, was set up in order to evaluate the effect of preparation procedure on NPs features and at the same time to broaden the nanoparticulate material encapsulation capability to drugs with different solubility properties.
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NPs sizes was slightly increased respect to plain PLGA NPs (Table 1, # 1-PLGA); no significant difference was observed in terms of Zeta potential. For both batches the process yield was about 40 % w/w.
Characterization of NPs in terms of size, surface charge and process yield. NPs size *Batch #
Zeta potential
Process yield (%, w/w)
Solvent nm±S.D.
PI
(mV)
1 - PLGA
DMSO
143.7±54.5
0.150
-17.7±1.3
44.28±2.6
2 - FQPV-PLGA
DMSO
202.3±73.8
0.133
-17.7±0.9
40.36±4.9
3 - PLGA
DCM/ Acetone
159.6±25.3
0.165
-18.3±1.0
40.63±5.3
4 - FQPV-PLGA
DCM/ Acetone
234.4±59.4
0.199
-20.3±1.8
41.25±6.1
*Each batch was prepared at least in triplicate.
Smart Biodegradable Nanoparticulate Materials
Current Nanoscience, 2016, Vol. 12, No. 3
parameters used in the preparation of batch #1 and #2 are not suitable for preparing a colloidal system in the nanometer scale, since the size of particles is about 2 m. The same situation occurred when factor X2 was kept at the lowest value (-1: PVA 0.2%). Consequently, batches prepared with the highest value of both X1 (80% v/v of Acetone) and X2 (PVA solution 1% w/w) showed dimension suitable to our purpose (batch #6 and #7;
