Int J App Pharm, Vol 12, Issue 6, 2020, 65-68Original Article
SELECTION OF THE COMPOSITION OF A LIPOSOMAL DOSAGE FORM OF A RUSSIAN SOMATOSTATIN ANALOGUE WITH ANTITUMOR ACTIVITY
MARIA DMITRIEVA1*, ZOYA SHPRAKH1,2, OLGA ORLOVA1, ELENA IGNATYEVA1, ANNA LANTSOVA1, LUDMILA NIKOLAEVA1,2, IVAN KRASNYUK2
1N. N. Blokhin National Medical Research Center of Oncology, Moscow, Russia, 2Sechenov University, Moscow, Russia
Email: dmitrieva.m@ronc.ru
Received: 28 Jul 2020, Revised and Accepted: 08 Sep 2020
ABSTRACT
Objective: Was to create the composition of the liposomal pharmaceutical form for injections of somatostatin analogue cyphetrylin using soybean phosphatidylcholine (SPC).
Methods: The cyphetrylin, active pharmaceutical ingredient (API), developed in the Chemical Synthesis Laboratory, the N. N. Blokhin National Medical Research Center of Oncology of the Ministry of Health of the Russian Federation; SPC and polyethylene glycol-2000-distearoylphosphatidylethanolamine (PEG-DSPE, Lipoid, Germany); cholesterol ≥99% (Sigma-Aldrich, Japan). The lipid film hydration method with subsequent liposomal dispersion filtration/extrusion through nylon membrane filters was used for the phospholipid vesicle production. Based on API and lipid components in different molar ratios, we studied over 15 model liposomal compositions and assessed each lipid's impact in use on quality attributes of resulting dispersions. Derived model samples of liposomal dispersion were estimated in terms of quality and efficiency of cyphetrylin encapsulation into vesicles, their average size and the surface charge (zeta potential), polydispersity index (PDI) and dispersion viscosity. We used spectral photometry, dispersion laser spectroscopy, electrophoretic particle mobility assay, and viscometry to assess these features.
Results: Pharmaceutical form components' desirable molar ratios determined: cyphetrylin/SPC at 1:60.0 and SPC/cholesterol/PEG-DSPE at 1:0.2:0.004, were determined. This composition allows cyphetrylin liposomal dispersion production with relatively stable vesicles of uniform size, 176 nm in diameter, and a 100% maximum rate of API encapsulation into the bilayer.
Conclusion: Technological and chemical/pharmaceutical studies resulted in selecting a preferable composition of an injectable liposomal pharmaceutical form model of somatostatin analog-based on the SPC.
Keywords: Somatostatin analogue, Liposomes, Soybean phosphatidylcholine, Pharmaceutical dosage form, Composition, The molar ratio
© 2020 The Authors. Published by Innovare Academic Sciences Pvt Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/)
DOI: http://dx.doi.org/10.22159/ijap.2020v12i6.39253. Journal homepage: https://innovareacademics.in/journals/index.php/ijap
INTRODUCTION
The concept of the liposomal system of active pharmaceutical ingredients (API) delivery revolutionized the pharmaceutical market. Liposomes, initially called “bangosomes”, were discovered and first described in 1965 by the British biophysicist Alec Bangham and his colleagues at Babraham Institute in Cambridge. Since then, liposomes have evolved from a simple cellular membrane model into an actively studied and applied to a delivery system for API of a different origin. So far, the FDA has authorized the clinical use of 15 liposomal substances in various medicine fields: oncology, dermatology, anesthesiology, and treatment of infectious diseases. All these medicinal products resemble free API for their main clinical effect. However, their toxicological criteria compare favourably, which improves the tolerance of these medicinal products, the costs of eliminating the adverse toxic events, and, consequently, the patient's quality of life. Besides, ca. 30 liposome-based developments, mostly intended to treat cancers, are now in the clinical trials pipeline [1−3].
There are no alternative delivery systems with all properties inherent in liposomes now. These nanostructures are biocompatible and biodegradable, which allows for their parenteral administration. Liposomes protect the surrounding body tissues from API cytotoxic action, fortifying the active substance against enzymatic destruction and extending the effect of API introduced into the body, and are capable of targeting delivery of API to organs, tissues, and individual cells. The liposomal delivery system allows for the encapsulation of both water-retaining and water-repellent substances, facilitating the production of their water-soluble forms and thus improving their bioavailability. It is essential because the amount of water-insoluble and not readily water-soluble in API has increased recently [4−6].
We counted approximately 75% new candidate substances for therapeutic product development, which are not readily water-soluble; most are antitumor agents. Poor water solubility is the integral property of many antitumor drugs for two main reasons. Firstly, candidate drugs' physical and chemical properties rarely focus on the screening of substances for antitumor activity. Secondly, to achieve permeability, activity, and stability, an antitumor agent must contain certain hydrophobic structural features that make API not freely water-soluble [7].
The Chemical Synthesis Laboratory of N. N. Blokhin National Medical Research Center of Oncology of the Ministry of Health of the Russian Federation derived a hydrophobic somatostatin analogue, cyphetrylin, with antitumor activity. Unlike other somatostatin equivalents, cyphetrylin is a non-cyclical peptide derived with classical peptide chemistry methods. By its chemical structure, cyphetrylin is a methyl ether of Nα-tret-butyloxycarbonyl-S-tetrahydropyranyl, cysteine phenyl, alanyl-D-tryptophyl-Nε-carbobenzoxylysyl threonine (fig. 1). All lateral reactive groups in its molecule are protected with tert-butyloxycarbonyl, benzyloxycarbonyl, and tetrahydropynal groups. In vivo studies, cyphetrylin inhibits the secretion of growth hormone, prolactin and insulin secretion. Cyphetrylin antitumor activity shown in continuous cell lines of mouse tumors: adenocarcinoma of the breast Ca755, human breast cancer BrC1, cervical cancer CC5, Lewis lung carcinoma LLC, and those of rat tumors: prostatic adenocarcinoma R-3327-H and DMBA-induced breast tumors [8−10].
Fig. 1: Cyphetrylin structural formula. Legend: R–tret-butyloxycarbonyl, R1–tetrahydropyranyl, R2–carbobenzyloxycarbonyl, R3–methyl
With the view to hydrophobic properties of cyphetrylin, the Pharmaceutical Form Development Laboratory of N. N. Blokhin National Medical Research Center of Oncology of the Ministry of Health of Russian Federation derived cyphetrylin a solid pharmaceutical dosage form of tablets, which has already passed Stage I clinical trials in patients with neuroendocrine tumors [11−14]. It was proposed to develop the liposome-based injectable pharmaceutical form as an alternative to cyphetrylin tablets, to improve its efficacy and mitigate adverse events.
MATERIALS AND METHODS
Мaterials
Cyphetrylin substance (N. N. Blokhin National Medical Research Center of Oncology of the Ministry of Health of Russian Federation), soybean phosphatidylcholine (SPC, Lipoid, Germany), cholesterol ≥99% (Sigma-Aldrich, Japan), polyethylene glycol-2000-distearoylphosphatidylethanolamine (PEG-DSPE, Lipoid, Germany), chloroform stabilized with ethanol (Сhimmed, Russia), 95% ethanol (Flora Kavkaza OSJC, Russia), water for injections; 1.2 um, 0.45 um and 0.22 um Pall nylon membrane filters, N66, d 25 mm (Pall Eurasia LLC, Russia).
Мethods
Production of liposomes with cyphetrylin
To produce cyphetrylin liposomes, we used the Bangham method [15]. Cyphetrylin substance and lipid components (SPC, cholesterol, and PEG-DSPE) were dissolved in chloroform. We transferred the resulting solution into a round-bottom flask and removed the organic solvents by vacuuming a Heidolph Hei-VAP Advantage rotary evaporator (Heidolph, Germany) at low-pressure at+(40±1) °С water bath until the film became evenly distributed on the formed flask walls. The lipid film was terminally dried in a vacuum (120−125 mbar) until the organic solvent residues were removed and then hydrated with water for injection to produce large multi-layer liposomes dispersion with the theoretical active substance concentration of 1.0 mg/ml. To reduce cyphetrylin liposomes size, the dispersion was successively screened through 1.2 um, 0.45 um, and 0.22 um nylon membranes in the LipexTM extruder (Northern Lipids, Canada). “Empty” liposomes were generated in the same way as those with an active substance, but without adding cyphetrylin to the drug components organic solution. Using cyphetrylin, SPC, cholesterol, and PEG-DSPE, we generated and tested 15 model compositions of cyphetrylin in the liposomal pharmaceutical dosage form with different components molar ratios (table 1) [16].
Table 1: Model compositions of the liposomal dosage form of cyphetrylin
| Formula | Molar ratio | |
| Cyphetrylin/SPC | SPC/cholesterol/PEG-DSPE | |
| F1 | 1:70.0 | 1:0.2:0.004 |
| F2 | 1:70.0 | 1:0.1:0.004 |
| F3 | 1:67.5 | 1:0.2:0.004 |
| F4 | 1:67.5 | 1:0.1:0.004 |
| F5 | 1:65.0 | 1:0.3:0.004 |
| F6 | 1:65.0 | 1:0.2:0.004 |
| F7 | 1:65.0 | 1:0.1:0.004 |
| F8 | 1:60.0 | 1:0.3:0.004 |
| F9 | 1:60.0 | 1:0.2:0.005 |
| F10 | 1:60.0 | 1:0.2:0.004 |
| F11 | 1:60.0 | 1:0.2:0.003 |
| F12 | 1:57.5 | 1:0.3:0.004 |
| F13 | 1:57.5 | 1:0.2:0.004 |
| F14 | 1:55.0 | 1:0.3:0.004 |
| F15 | 1:55.0 | 1:0.2:0.004 |
Сyphetrylin inclusion in liposomes
Cyphetrylin content in liposomes was performed using the working reference standard (RS) at 282±2 nm wavelength in Cary 100 spectrophotometer (Agilent Technologies, Australia). As liposomes lipid components absorb in this spectrum, too, measurements were conducted in comparison with the “empty” liposomes solution.
Cyphetrylin RS preparation: add about 15 ml 95% ethanol to 2.5 mg cyphetrylin. The resulting ethanol solution is transferred into a 25 ml volumetric flask and diluted with ethanol. The solution to be used when fresh.
Comparison solution preparation: 2 ml liposomal dispersion of “empty” liposomes is dissolved with ethanol in a 25 ml volumetric flask and diluted to the volume with ethanol. The solution is used when fresh.
Test solution preparation: dissolve 2 ml cyphetrylin liposomal dispersion into a 25 ml volumetric flask. Add a small quantity of 95% alcohol. Mix and dilute to the volume with alcohol.
Measure the test solution's optical density in trays with 10 mm optical layer “thickness”, in maximum absorption compared with the comparison solution. Simultaneously, measure the optical density of the cyphetrylin RS solution vs that of the comparison solution. Using the formula (the dilution is identical), calculate the cyphetrylin concentration in liposomes (Х, mg/ml):
А and А0–optical densities of the liposomal sample solutions and cyphetrylin RS, respectively; а0–weight of cyphetrylin RS, mg; 2–volume of cyphetrylin liposomal sample, ml [16].
Determination of cyphetrylin liposomal encapsulation efficiency
As cyphetrylin is a hydrophobic substance and directly included in the lipid bilayer during liposome production, the encapsulation efficiency (EE) was determined as the ratio of API concentration in post-extrusion liposomal dispersion to cyphetrylin concentration in the dispersion after the lipid film hydration. The indicator expressed as a percentage:
Сe–cyphetrylin concentration in dispersion after extrusion, mg/ml; Сh–cyphetrylin concentration in dispersion after hydration, mg/ml [16].
Determination of average diameter, polydispersity index (PDI) and the zeta potential for cyphetrylin liposomes
The liposomal test by size and PDI was conducted using the correlation light scattering spectroscopy. Zeta potential of vesicles was determined by measuring their electrophoretic mobility. All measurements used zeta sizer Nano-ZS 3600 (Malvern, U. K.). 100 μl test sample of freshly made liposomal dispersion was measured with an automatic pipette, poured into a 100 ml volumetric flask, and diluted in volume with water for injection. A 1 ml diluted sample was transferred to a polystyrene tray, placed into the zetasizer cell, and the above indicators were measured [17, 18].
Determination for cyphetrylin liposomal dispersion viscosity
Dynamic viscosity of cyphetrylin liposomal dispersion samples was determined using Vibro Viscometer SV-10 (AND Company Limited, Japan). All measurements were conducted at a temperature ranging from 21 °С to 25 °С. To this end, 10 ml of liposomal dispersion was poured onto the device's tray and measured [19].
RESULTS AND DISCUSSION
In 2014−2017, the N. N. Blokhin National Medical Research Center of Oncology of the Ministry of Health of the Russian Federation researched on the development of a liposomal drug form of cyphetrylin. The components for cyphetrylin liposomal pharmaceutical dosage form were selected based on their functional purpose. Phosphatidylcholine of animal origin, derived from egg yolk (EPC), was used as the principal liposomal bilayer-forming excipient because of its properties. It has an acceptable phase-transition temperature (+37 °С), good solubility in organic solvents, biocompatibility and natural origin. Natural phospholipids are preferred in pharmaceutical technology compared with their synthetic counterparts because they are available on a large scale, at a reproducible quality with low production costs, and manufactured with smaller quantities of chemical agents and solvents a higher yield of the finished product [20]. To enhance the bilayer rigidity and improve the phospholipid vesicle stability in the blood flow, cholesterol was added to liposomes. To protect the vesicles from being captured by reticuloendothelial cells after their introduction into the body and to slow down their clearance from the blood flow, we developed the stealth-liposomes with cyphetrylin. To this end; we injected the phospholipid “sewed” with PEG-DSPE into the bilipid layer of the pharmaceutical form, which increases osmotic pressure near the liposomes and prevents their discovery by macrophages. Using these components, we derived the model of the liposomal pharmaceutical dosage form with molar ratios of cyphetrylin/EPC at 1:70.0 and those of EPC/cholesterol/PEG-DSPE at 1:0.2:0.003. The created cyphetrylin liposomal dispersion has 98% drug encapsulation rate and with 168±6 nm vesicle size [16].
To improve the injectable liposomal form of cyphetrylin, it was proposed to modify cyphetrylin liposomal composition by replacing EPC to SPC. Egg lecithin contains less unsaturated lipids than soybean lecithin (table 2) [19]. However, by forming different bonds with phospholipids, the hydrophobic active substance is encapsulated into the lipid bilayer. Based on this data, we suggested that the phosphatidylcholine (PC) origin may significantly impact the API encapsulation rate and that a large number of unsaturated bonds would lead to a lower lipid concentration in the drug and a higher API/PC ratio.
Table 2: Egg and soybean lecithin lipid composition
| Chain composition | Egg lecithin | Soybean lecithin |
| 16:0 | 32 (33) | 15 (14) |
| 16:1 | 5 (1) | 4 |
| 16:2 | 1 | 0 |
| 18:0 | 14 (13) | 1 (4) |
| 18:1 | 19 (32) | 14 (11) |
| 18:2 | 13 (16) | 59 (64) |
| 20:1 | 4 | 0 |
| 20:2 | 6 | 0 |
| 20:3 | 2 | 1 |
| 20:4 | 2 (3) | 4 |
| other | 2 (5) | 2 (3) |
| saturated | 46 | 16 |
| unsaturated | 52 | 82 |
The test was conducted by reducing SPC content in cyphetrylin/SPC from the initial 1:70.0 established for liposomes with EPC, as described above, and by varying the lipid composition ratio, with monitoring of such indicators as EE, average post-extrusion vesicle size and their fraction distribution index (PDI), zeta potential of liposomes and the resulting dispersion viscosity (table 3).
Table 3: Characteristics of SPC-based investigatory compositions of cyphetrylin liposomal pharmaceutical form
| No | EE, % | Vesicle size, nm | PDI | Zeta potential, mV | Viscosity, mPa·s |
| F1 | 100.0±0.62 | 171±6.94 | 0.170±0.01 | -13.2±0.75 | 9.8±1.19 |
| F2 | 100.0±1.51 | 167±6.05 | 0.282±0.01 | -19.3±1.31 | 8.4±1.24 |
| F3 | 100.0±0.59 | 179±7.11 | 0.231±0.02 | -16.4±1.10 | 5.6±0.33 |
| F4 | 99.0±0.82 | 173±7.01 | 0.177±0.01 | -14.6±0.77 | 8.8±0.50 |
| F5 | 100.0±0.70 | 173±6.50 | 0.290±0.01 | -18.1±0.95 | 10.8±0.62 |
| F6 | 100.0±0.33 | 174±5.91 | 0.195±0.01 | -19.6±0.92 | 8.5±1.01 |
| F7 | 100.0±0.73 | 210±8.22 | 0.213±0.01 | -19.3±1.02 | 6.3±0.74 |
| F8 | 100.0±0.97 | 174±7.15 | 0.230±0.01 | -20.0±1.07 | 6.7±0.55 |
| F9 | 99.6±0.80 | 170±7.23 | 0.168±0.01 | -19.2±0.72 | 10.7±0.43 |
| F10 | 100.0±0.54 | 176±6.02 | 0.212±0.01 | -18.3±0.90 | 7.5±0.75 |
| F11 | 99.0±0.50 | 183±6.55 | 0.256±0.02 | -18.0±1.25 | 6.5±0.67 |
| F12 | 95.6±0.88 | 185±7.30/11±0.02 | 0.195±0.01 | -17.5±1.18 | 7.9±0.35 |
| F13 | 90.8±1.23 | 211±7.89 | 0.162±0.01 | -17.3±1.30 | 6.2±0.49 |
| F14 | 93.1±1.04 | 152±6.70/11±0.10 | 0.099±0.01 | -18.0±0.95 | 8.6±0.58 |
| F15 | 88.1±1.10 | 182±8.05 | 0.110±0.01 | -19.7±0.85 | 6.6±0.71 |
Note: Data given in mean±SD, n=3
As a result, we established that the content of all lipid components influenced cyphetrylin liposomal EE. When the overall lipid concentration decreased, the 100% maximum encapsulation was achieved in a liposomal composition No. 10 with the molar ratio of cyphetrylin/SPC at 1:60.0 and those of SPC/cholesterol/PEG-DSPE at 1:0.2:0.004. Further increase in cyphetrylin/SPC ratio resulted in a significant reduction of the encapsulation rate to ~91%, which may have increased to 95.6% at a higher cholesterol content (F12). A more significant increase in its concentration is not appropriate due to the deterioration of the dispersion technological properties resulting from the rigid liposomal structure. We also noted that both the decrease and the increase in pegylated phospholipid content contribute to a slight reduction in cyphetrylin vesicular EE.
When reviewing other quality parameters, we did not establish any correlation between their values and the liposomal lipid composition. The size distribution curves review suggested that virtually all model liposomes' diameter did not exceed the preferable one for therapeutic use, 220 nm, and PDI are below 0.3, which testifies to the narrow size distribution of the particles [21]. The only exception is being F8: the vesicle diameter amounted to 243 nm. F12 and F14 displayed the bimodal size distribution, which points to two vesicle fractions with different sizes. Vesicle size reduction as the cholesterol level increases may be accounted for by a higher packing density. The electrophoretic mobility measurement analysis showed that cyphetrylin liposomes charge of the prepared compositions ranges from-13.2 mV to-20.0 mV, and dispersion dynamic viscosity range from 5.6−10.8 mPa·s.
The comparative data analysis by quality attributes of the investigated compositions allowed to select the preferable model for the production of cyphetrylin liposomal pharmaceutical dosage form based on soybean lecithin with the following molar ratios of components: cyphetrylin/SPC at 1:60.0 and SPC/cholesterol/PEG-DSPE at 1:0.2:0.004.
The conducted study suggests that phospholipids derived from different sources may influence active hydrophobic substance loading into a liposomal membrane. If cyphetrylin was included, the SPC selected as the primary, bilayer-forming component in liposomal pharmaceutical dosage form for further research had the advantage.
CONCLUSION
Cyphetrylin, the hypothalamic hormone somatostatin analogue with antitumor activity, is a promising drug to treat many cancers, including neuroendocrine tumors. Cyphetrylin liposomal encapsulation was proposed for the injectable pharmaceutical dosage form creation to overcome the active substance's low water solubility. The conducted research helped select the desirable model composition of the cyphetrylin SPC-based liposomal pharmaceutical dosage form.
FUNDING
Nil
AUTHORS CONTRIBUTIONS
Maria Dmitrieva: Research design, obtaining and analyzing data, writing the text of the manuscript. Zoya Shprakh: Research design, preparation of the manuscript, translation. Olga Orlova, Ivan Krasnyuk: Research design, data analysis, verification of the final version of the manuscript. Elena Ignatyeva: Obtaining and analyzing data. Anna Lantsova, Ludmila Nikolaeva: Review of publications, design of the manuscript.
CONFLICT OF INTERESTS
The authors declare no conflict of interest
REFERENСES
Bulbake U, Doppalapudi S, Kommineni N, Khan W. Liposomal formulations in clinical use: an updated review. Pharmaceutics 2017;2:12.
Bangham AD. Liposomes: the babraham connection. Chem Phys Lipids 1993;1−3:275−85.
Svistelnik AV, Khanin AL. Liposomal drugs: opportunities and prospects. Med Kuzbass 2014;2:7−16.
Lee MK. Liposomes for enhanced bioavailability of water-insoluble drugs: in vivo evidence and recent approaches. Pharmaceutics 2020;3:264.
Lee Y, Thompson DH. Stimuli-responsive liposomes for drug delivery. Wiley Interdiscip Rev Nanomed Nanobiotechnol 2017;5:10.
Khan YY, Suvarna V. Liposomes containing phytochemicals for cancer treatment−an update. Int J Curr Pharm Res 2017;1:20−4.
Gala UH, Miller DA, Williams RO. Harnessing the therapeutic potential of anticancer drugs through amorphous solid dispersions. Biochim Biophys Acta Rev Cancer 2020;1:188319.
Smirnova AP, Sushinina LP, Ustinkina SV, Shprakh ZS, Budko AP, Smirnova LI, et al. Synthesis and antitumor activity cifetrilin when administered orally. Russ J Biother 2009;2:18.
Shprakh ZS, Yartseva IV, Ignateva EV, Smirnova AP, Sushinina LP, Ustinkina SV, et al. Synthesis and chemico-pharmaceutical characteristics of somatostatin analog with antitumor activity. Pharm Chem J 2014;3:159−62.
Kubasova IYu, Borisova LM, Kiseleva MP, Smirnova LI, Smirnova AP, Ustinkina SV, et al. Search of potential antitumour compounds among of somatostatin hypothalamic hormone analogues. Russ J Biother 2006;3:128–33.
Shprakh Z. Somatostatin analogues for the treatment of neuroendocrine tumours. Dosage forms and routes of administration (review). Int J Appl Pharm 2020;12:6−11.
Mikhaevich EI, Yavorskaya NP, Golubeva IS, Polozkova AP, Partolina SA, Oborotova NA. Studying the possibility of oral delivery of cifetrilin. Russ J Biother 2012;1:3–8.
Shprakh Z, Orlova O, Ignatieva E, Oborotova N, Bunyatyan N. Formulation and evaluation of somatostatin analogue tablets. Int J Appl Pharm 2019;4:220−3.
Shprakh Z, Borisova LM, Kiseleva MP, Smirnova ZS. Preclinical study of cyphetrylin antitumor efficiency on experimental animal tumors. Exp Clin Pharmacol 2019;8:27−31.
Singha Roy A, Das S, Samanta A. Design, formulation and evaluation of liposome containing Isoniazid. Int J Appl Pharm 2018;10:52−6.
Sanarova EV, Zhang Xi, Dmitrieva MV, Lantsova AV, Orlova OL, Polozkova AP, et al. Features of the technology of liposomal formulation of a analog hypothalamic hormone somatostatin. Russ J Biother 2016;4:78–84.
Mehanna M, Elmaradny H, Samaha M. Mucoadhesive liposomes as ocular delivery system: physical, microbiological, and in vivo assessment. Drug Dev Ind Pharm 2010;36:108−18.
Zhou T, Tang X, Zhang W, Feng J, Wu W. Preparation and in vitro and in vivo evaluations of 10-hydroxycamptothecin liposomes modified with stearyl glycyrrhizinate. Drug Delivery 2019;1:673−9.
Budai L, Kaszas N, Grof P, Lenti K, Maghami K, Antal I, et al. Liposomes for topical use: a Physico-chemical comparison of vesicles prepared from egg or soy lecithin. Sci Pharm 2013;4:1151−66.
Van Hoogevest P, Wendel A. The use of natural and synthetic phospholipids as pharmaceutical excipients. Eur J Lipid Sci Technol 2014;9:1088−107.
Zweers ML, Grijpma DW, Engbers GH, Feijen J. The preparation of monodisperse biodegradable polyester nanoparticles with a controlled size. J Biomed Mater Res B Appl Biomater 2003;66:559–66.