Drug Delivery Systems Based on Hydroxyethyl Starch - American

Subscriber access provided by Fudan University

Review

Drug Delivery Systems Based on Hydroxyethyl Starch Constantinos M. Paleos, Zili Sideratou, and Dimitris Tsiourvas Bioconjugate Chem., Just Accepted Manuscript • Publication Date (Web): 21 Apr 2017 Downloaded from http://pubs.acs.org on April 23, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Bioconjugate Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 39 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Bioconjugate Chemistry

Drug Delivery Systems Based on Hydroxyethyl Starch Constantinos M. Paleos†,‡,* Zili Sideratou† and Dimitris Tsiourvas† †

NCSR “Demokritos”, Institute of Nanoscience and Nanotechnology, 15310 Aghia

Paraskevi, Attiki, Greece. ‡

Regulon AE, Apollonos 1, 19400 Koropi, Attiki, Greece.

* To whom correspondence should be addressed. Phone: +30-10-6503666, FAX: +30-210-6511766, e-mail: [email protected]

Abstract Τhe advantageous biological properties of hydroxyethyl starch (HES) triggered research interest towards design and synthesis of Drug Delivery Systems (DDSs) based on this polysaccharide. Convenient reaction schemes, including one-step reactions, led to the synthesis of HES conjugates with selected anticancer molecules or therapeutic proteins. Nanocapsules and hydrogels based on HES were also prepared and studied as prospective drug delivery systems. Formulations originating from these drug conjugates and also from nanocapsules and hydrogels loaded with drugs were characterized, highlighting on the extension of their half-life in plasma which is a critical property as far as their efficacy is concerned. Results obtained in vitro and in vivo proved promising, justifying additional experiments to be undertaken with such systems including their multifunctionalization. These promising formulations that are discussed in this topical review is expected to further increase interest in applying HES for molecular constructing novel DDSs with enhanced efficacy which may, in the future, find clinical applications.

ACS Paragon Plus Environment

Bioconjugate Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1. Introduction A literature investigation on drug delivery research has shown that extensive scientific and technological work has been performed in the last fifty years starting already from 1965, when Bangham1 prepared for the first time liposomes, and in the mid-seventies when Ringsdorf2 proposed the attachment of pharmacologically active agents to water soluble polymers. Ever since, a great volume research has been contacted in the domain of drug delivery systems development, aiming to systems with enhanced specificity and efficacy. Among the water-soluble polymers investigated as drug delivery systems (DDSs), polysaccharides have extensively been investigated,3-6 due to the abundance of functional groups ready for modification, wide availability as well as biocompatibility, low immunogenicity and biodegradability in biological media. Hydroxyethyl Starch, (HES),7-9 is a semisynthetic polysaccharide prepared by interacting starch with ethylene oxide in alkaline media. It is a flexible and functional polymer consisting of alpha (1,4)glycosidic-linked anhydroglucose units while chain branching results from (1,6)glycosidic bonds. The structure of HES, Scheme I, is similar to glycogen, i.e. the branched glucose storage human polysaccharide. The non-immunogenicity of HES is possibly attributed to the common structural features between glycogen and HES. Furthermore, HES is more soluble than starch in water, and also exhibits higher stability to hydrolysis. Most interestingly an increase of half-life is observed in vivo.10 The halflife for starch is a few minutes, due to its hydrolysis by serum amylases,11 but it is significantly enhanced for HES due to steric hindrance of the hydroxyethyl moieties. In addition, the biodegradability of HES can be controlled since the rate and degree of degradation can be fine-tuned by modifying the molar mass and the degree of hydroxyethylation.12 These structural features of HES have established this polymer as a first line colloidal Plasma Volume Expander,13 although safety concerns have arisen for increased risk of death and kidney injury when administered to critically ill patients requiring fluid resuscitation.14 It has however to be noted that in DDSs experiments smaller concentrations than those employed in plasma volume expander applications are used and therefore such a safety problem is avoided. Due to its properties, it was utilized


Page 2 of 39

Page 3 of 39 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


for the synthesis of selected DDSs that could overcome hurdles during delivery and hopefully pave the way to successful clinical studies. To this end, conjugates of HES with well-established anticancer compounds and therapeutic proteins have been prepared and characterized. In addition, HES-based nanocapsules and hydrogels were prepared and loaded with model drugs to be also applied as prospective drug delivery systems. Formulations derived from HES-anticancer compound conjugates, HES-therapeutic protein conjugates and also HES-based nanocapsules and hydrogels will be presented. Also, due to its polyhydroxylated nature, its high hydrophilicity and high flexibility, it has been investigated as a substitute of poly(ethylene glycol) (PEG) in drug delivery. The results are promising and it is anticipated that these first reports, will lead to further studies on the application of HES in new drug formulations of enhanced effectiveness.

0 Scheme I: A portion of the chemical structure of HES.

2. Covalent and Non-covalent Binding of Selected Anticancer Drugs to HES A common strategy was adopted for the synthesis of conjugates between anticancer drugs and HES. Specifically, HES of appropriate number average molecular weight (Mn) and degree of substitution with hydroxyethyl groups (Ms), or a functionalized derivative of this polymer, interacts either with an anticancer drug or with an appropriate derivative, affording the corresponding conjugate. Because of this slight modification, the favorable properties of HES mentioned above, remain more or less intact in the final system. Some



anticancer drug formulations based on HES will be critically and comparatively reviewed with the corresponding non-HESylated ones in the following 2.1 to 2.6 subchapters. 2.1 Non-covalent Binding of HES to Doxorubicin Due to the polyhydroxylated character of HES (Mn 115-140 kDa, Ms=0.40-0,044) it was feasible to react with succinic anhydride introducing carboxylic acid groups in the polymer through half-esterification. Thus its carboxylated derivative C-HES15 was obtained. The carboxylic groups subsequently can electrostatically interact with the amino group of doxorubicin (DOX) yielding the corresponding salts C-HES-DOX. In this case doxorubicin,16 which is widely used in chemotherapy for the treatment of various tumors including hematological malignancies as well as for many types of carcinoma and soft tissue sarcomas, is non-covalently bound to HES. This may be assisted by the presence of nanocavities of the branched HES derivative, in an analogous manner with the non-covalent encapsulation of drugs inside the nanocavities of synthetic hyperbranched polymers.17 The resulting nanoparticles had sizes of 25±3 nm in diameter, as determined with dynamic light scattering spectroscopy, and DOX loading of ~0.18 gram per gram of C-HES, as determined employing UV–Vis spectroscopy. Representative images of fluorescence microscopy on DU145 cells incubated with DOX and C-HES-DOX are shown in Figure 1. Cells incubated with free DOX (Figure 1A) show DOX-fluorescence inside the nuclei, i.e. at the location where DOX is acting,18 while cells incubated with C-HES-DOX exhibit intense DOX-fluorescence within the cytosol. This was attributed to DOX encapsulation in C-HES which apparently has a different sub-cellular localization compared to free DOX.

Figure 1. DU145 cells incubated with (A) free DOX and (B) C-HES-DOX for 3h; DOX concentration 2 µΜ (reprinted from ref. 15).


Page 4 of 39

Page 5 of 39 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


The toxicity of C-HES-DOX vs free DOX is depicted in Figure 2 in which free DOX showed higher toxicity. However, after a period of 48h, C-HES-DOX was almost equally effective to free DOX as far as cytotoxicity is concerned. It must also be emphasized that C-HES has no notable toxicity at all investigated concentrations. Despite the fact that CHES-DOX is located inside cell cytosol, it is apparent that in the elapsed period of time, controlled DOX release from the complex C-HES-DOX is taking place. This results in increased cell toxicity, which almost reaches the toxicity of free DOX within 48 h. Also, the intense C-HES-DOX fluorescence which indicates enhanced DOX localization within the cell, was attributed to increased cellular uptake of the polysaccharide.

Figure 2. Comparative cytotoxicity of C-HES, free DOX, and C-HES-DOX following 3h incubation. In all experiments, DOX concentration was 1, 5, and 10 µM while the corresponding C-HES concentration was 3.4, 17.0, and 34.0 µg mL-1, respectively. The cytotoxicity was assessed at 24 and 48 h post-incubation by standard MTT assays (reprinted from ref. 15).

2.2

Covalent HES - Doxorubicin Conjugates

Following a different approach,19 HES (Mn 130 kDa, Ms 0.4) was conjugated to doxorubicin through a pH-sensitive hydrazine bond, producing HES-Hyd-DOX, 1. To assess the efficacy of this conjugate as a chemotherapeutic agent, a closely similar ester conjugate, HES-SAD, 2, which is pH–insensitive, was also synthesized. The release profiles of the two polymers at various pH values differ as shown in Figure 3, which was



Page 6 of 39

attributed to the acid-sensitive linker between HES and DOX. This pH-sensitive behavior may have a significant impact on the anticancer efficacy of HES-Hyd-DOX.

HO

HO

O O O

O

HO

OH

OH

HO HO

O

O

O

O

OH

OH HO

O

OH HO

O

NH

O

OH

O

OH OH

O

OH

O

OH

O

N OH

O

OH

OH

OH HO

O

O

OH

O

O

O

O

OH

OH OH

HO

O

O

O

O

OH

HO

O

O

O

O

OH

O

O

HO

O

OH

OH

HO

OH

O

O

OH

OH

O HO

O

O O

N H

O

O

OH

NH2 O

O

OH

1, HES-Hyd-DOX

HO

HO

O

2, HES-SAD

Figure 3. In vitro DOX release profiles for HES-Hyd-DOX (A) and HES-SAD (B) conjugates in PBS at 37 oC at pH 4.0, 5.3, 6.8 and 7.4 (reprinted from ref. 19).


Page 7 of 39 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


The cellular uptake and intracellular drug release of the conjugates were investigated by confocal laser scanning microscopy (CLSM) and flow cytometry. Thus, HES conjugates were incubated with HepG2 cells for 2 or 6 hours. Compared to HES-SAD the pH-sensitive HES-Hyd-DOX conjugate exhibited increased intracellular fluorescence as the incubation period had increased from 2 to 6 hours, Figure 4B. In contrast, no difference in fluorescence intensity was observed for the insensitive HES-SAD derivative (Figure 4) in analogous experiments.

Figure 4. Representative CLSM images of HepG2 cells incubated with HES-SAD for 2 h (A) or for 6 h (B) and incubated with HES-Hyd-DOX for 2 h (C) or for 6 h (D). In each row the images show a differential interference contrast (DIC) image, cell nuclei (blue, stained with DAPI), DOX localization in cells (red), and overlays of the three images (reprinted from ref. 19).



In addition the in vitro cellular proliferation inhibition of HES-Hyd-DOX (two different derivatives i.e HES-Hyd-DOX1 with 19% DOX content and HES-Hyd-DOX2 with 26% DOX content), and HES-SAD (9 % DOX content), against HepG2 cells and HeLa cells, as determined by MTT assay, have revealed that DOX release was induced by the low pH values of the endosomal environment leading to enhanced inhibition of cell proliferation, Figure 5, for the pH-sensitive conjugates compared to the insensitive one. It appears therefore that a novel promising pH-triggered drug delivery system can be added to the list of effective doxorubicin formulations.


Page 8 of 39

Page 9 of 39 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 5. Cytotoxicity of DOX (control), HES-Hyd-DOX1 (19% DOX content), HESHyd-DOX2 (26 % DOX content), and HES-SAD (9 % DOX content), toward HepG2 cells after incubation for 24 h (A), 48 h (B) and 72 h (C) (reprinted from ref. 19).

2.3 Redox-Sensitive HES-SS-Doxorubicin Conjugate For assessing the effect of tumor microenvironment on the efficacy of HES-Doxorubicin conjugate (HES: Mn 200 kDa, Ms 0.5) a modified redox-sensitive covalent HES-SS-DOX conjugate, 3, was prepared20 having a diameter of 20 nm. Tumor targeting was attributed to the increased glutathione (GSH) levels in the tumor microenvironment. GSH is an important antioxidant present in both animals and plants and is able to reduce disulfide bonds found in the biological milieu to cysteines. Elevated levels of GSH are typically present within tumor cells, and this has been exploited as a means to induce triggered drug release. The non-redox-sensitive conjugate HES-DOX, 4, not bearing the central disulfide moiety was also prepared as control. HES-SS-DOX was relatively stable under extracellular GSH concentration (∼2 µM) while in contrast, it quickly released DOX intracellularly, due to the high intracellular GSH levels (2−10 mM).

HO

HO

O O

O O

OH

O

O OH

OH

HO

HO

HO

HO

O

O O

OH

OH

O

OH

OH

O

OH

OH

HO O

S

OH

O

O

OH

N H

O

OH

O O

O

O

OH

N H

O

OH

O

HO

HO

OH

O O

3, HES-SS-DOX

HO

HO

4, HES-DOX


O

OH OH

S O

O

O

OH

OH

HO

O

O

O

O

O

HO

O

O OH

OH

HO

O

O

O

OH

O

HO

O

O

OH

OH

HO

OH

O O

OH

O

O

O

O


In vitro cell and in vivo animal studies confirmed that HES-SS-DOX had increased GSH-mediated cytotoxicity, prolonged plasma half-life time and increased accumulation inside the tumor compared to free DOX. HES-SS-DOX showed better antitumor efficacy and reduced toxicity as compared to free DOX in the in vivo study employing male SD rats. Specifically, it has been found that the blood elimination half-life time of HES-SSDOX and HES-DOX increases 13.8 and 18.2 times, respectively. Also the tumor accumulation of HES-SS-DOX and HES-DOX is 3.3 and 3.5 times higher 48 h following injection and both acute and long-term toxicity were significantly reduced. In addition to GSH induced cytotoxicity, HES-SS-DOX exhibits the highest tumor inhibition rate (81.0 ± 6.9%) compared to those of the non-redox-sensitive HES-DOX (55.2 ± 24.3%) or of free DOX (72.4 ± 11.0%). These results reveal that conjugation with HES can alter the in vivo activity of DOX and reduce its heart and kidney cytotoxicity, emphasizing the significance of HES conjugation. It is also evident that the disulfide bond linkage between HES and DOX is critical for a superior in vivo antitumor activity, underlining the advantage of tumor microenvironment stimuli responsive polymer−drug conjugates. In conclusion, the redox-sensitive HES-SS-DOX conjugate exhibits a promising clinical potential. 2.4 Covalent HES–10-hydroxy Camptothecin conjugate The anticancer compound 10-hydroxy camptothecin (10-HCPT)21 is a camptothecin derivative that exerts strong cytotoxic and antitumor activity against gastric carcinona, leukemia head and neck tumors. The therapeutic applications of 10-HCPT are however inhibited because of its low water solubility and limited stability in vitro and in vivo.22 This derivative also exhibits a pH-dependent equilibrium between the lactone and carboxylate forms under physiological conditions. The lactone form is the antitumor agent, whereas the carboxylate form is inactive. In addition, the carboxylate form is associated with severe side effects. For this reason, increased water solubility, maintenance of the stability of the lactone form and increased half-life are required for enhancing the efficacy of 10-HCPT. In this study, 10-HCPT-HES conjugate, 5, was synthesized,21 through the modification of both HES and 10-HCPT. HES of Mn=130 kDa / Ms=0.4 and Mn=200 kDA / Ms=0.5 were employed. Succinic anhydride was used for the introduction of the


Page 10 of 39

Page 11 of 39 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


carboxyl groups into HES at various degrees of substitution, while the modification of 10-HCPT took place at the 20-hydroxyl group of 10-HCPT. The two 10-HCPT-HES conjugates, i.e. 10-HCTP-HES (130kDa/0.4) and 10-HCTP-HES (200 kDa/0.5), showed sustained release behavior in phosphate-buffered saline (PBS), rat plasma and liver homogenates. The release profiles of 10-HCTP for both conjugates in the various media tested are shown in Figure 6. It has to be noted that drug release was not complete after 48 h for 10-HCTP-HES conjugates, exhibiting therefore an apparent sustained release effect, which suggests prolonged circulation and favorable cancer treatment.

5, 10-HCTP-HES



Figure 6. Release of 10-HCPT from 10-HCPT–HES conjugates at 37 oC in PBS: (A) 10HCPT–HES conjugate (130 kDa/0.4); (B) 10-HCPT–HES conjugate (200 kDa/0.5), and in plasma and liver homogenate (C). 1: 10-HCPT–HES (130 kDa/0.4); 2: 10-HCPT–HES (200 kDa/0.5) (reprinted from ref. 21).

Cytotoxicity of 10-HCTP-HES conjugates, compared to those of free 10-HCPT as control, was investigated by employing Hep-38 and SMMC-7721 cell lines. In summary, 10-HCPT-HES conjugates exhibited higher cytotoxicity, compared to the free 10-HCPT, especially at low concentrations as shown in Figure 7. Insignificant cell cytotoxicity of free HES was observed towards the same cell lines. In vivo studies employing nude mice with Hep-3B tumor revealed that the bioavailability of the two HES conjugates were 30-fold and 40-fold higher compared to free 10-HCPT, while tumor growth after intravenous administration of the 10-HCPTHES conjugate at a dose of 1.0 mg/kg was inhibited by ~78% compared to ~31% inhibition for free 10-HCPT. These certainly encouraging results show the higher efficacy of HES conjugates both in vitro and in vivo.


Page 12 of 39

Page 13 of 39 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 7. Cell viability of Hep-3B and SMMC-7721 cancer cell lines after 48 or 72 h incubation with 10-HCPT–HES conjugates and free 10-HCPT. Conjugate 1: 10-HCPT– HES (130 kDa/0.4); conjugate 2: 10-HCPT–HES (200 kDa/0.5) (reprinted from ref. 21).

2.5 Covalent HES-5-Fluorouracil conjugate For developing a sustained-release drug delivery system with increased half-life, the widely used anticancer drug 5-fluorouracil (5-FU)23 was initially transformed to 5fluorouracil-1-acetic acid (FUAC). Subsequently this derivative was conjugated through esterification to HES, affording the HES-FUAC conjugate, 6.24 The content of FUAC in the conjugate was 15% (15 mg of FUAC per 100 mg of HES-FUAC conjugate) and was found dependent on the number of hydroxyl groups of HES and the reaction conditions employed. The conjugate was relatively stable in acidic media, i.e. at pH 5.8, slowly releasing FUAC, but it was more easily hydrolysable upon pH and temperature increase.



6, HES-FUAC

The conjugate was also hydrolysable in human and rat plasma releasing FUAC with half-lives of 20.4 h and 24.6, respectively. Both 5-FU and FUAC were, however, released in a rat homogenate following incubation for 12 h. The percentage of released 5-FU and FUAC metabolites obtained are shown in Figure 8. The pharmacokinetic behavior was comparatively investigated in rats following intravenous injection of 5-FU, FUAC and HES-FUAC. Upon administration of free 5-FUAC, its plasma concentration fell rapidly, while the FUAC concentration in plasma after administration of HES-FUAC showed a slower elimination. It was suggested that after intravenous injection of the HES-FUAC, the ester bonds located at or near the surface of HES immediately hydrolyze releasing FUAC. However, the FUAC-ester conjugated moieties located inside the polymer are released only when HES is hydrolyzed by plasma amylases. This significantly increases the half-life of FUAC in the blood circulation compared to that of free FUAC. Overall, drug release determined in vitro and in vivo shows that HES is a promising delivery system for the controlled-release of this anticancer drug.


Page 14 of 39

Page 15 of 39 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 8. Release profiles of FUAC from the HES-FUAC conjugate in human plasma (closed diamonds) or rat plasma (closed squares), as well as of FUAC (open diamonds) or 5-FU (open squares) in rat liver homogenate, at 37 oC (reprinted from ref. 24).

2.6 Covalent HES-Methotrexate conjugate The conjugate of methotrexate (MTX) to HES, HES-MTX, has also been prepared and characterized.25 Activation of the carboxyl group of MTX for binding covalently to HES (130/0.4) was performed by a well-known method.26 Following removal of the free MTX, the conjugate contained 52 x 10-3 covalently linked MTX residues per anhydroglucose unit. It was proposed that HES-MTX conjugates because of their hydrodynamic size (15 ± 6 nm, polydispersity index = 0.17) can avoid renal clearance. The surface of HES-MTX conjugates has a negative charge (~ -28 ± 8 mV) which, being similar to those on the vascular endothelial surface may result in a longer half-life in plasma and consequently increased tumor accumulation of this conjugate due to the enhanced permeability and retention (EPR) effect.27 In vitro studies were conducted employing various cell lines, while the antitumor efficacy in vivo was tested in NOD/SCID mice subcutaneously inoculated with MV-4-11 human leukemia cells and with CDF1 mice intraperitoneally inoculated with murine leukemia cells. The in vivo experiments showed much higher anticancer efficacy of HESMTX conjugates compared to non-conjugated ones. Specifically, leukaemia-bearing mice



treated with HES-MTX had significantly higher survival rate compared to animals either untreated or treated with free MTX. In addition, HES-MTX significantly inhibited the growth of MV-4-11 tumor bearing NOD/SCID mice compared to the two controls (Figure 9). The well-established therapeutic efficacy of MTX is therefore further enhanced when applied as a conjugate of HES which can lead to improved therapies.

Figure 9. Tumor growth kinetics and tumor growth inhibition parameters (TGI, inset) of MV-4-11 bearing NOD/SCID mice treated either with HES-MTX, or with free MTX. The control group received saline (reprinted from ref. 25).

3. HESylation of Proteins: Covalent Binding of HES to Proteins There has been lately increasing interest in strategies for formulating and delivering therapeutic proteins. It has been found that biotechnology drugs accounted for one fifth of all the blockbuster drugs as of 2008,28-31 despite the hurdles that have been encountered during their formulation and delivery. Several of these biopharmaceuticals, excluding monoclonal antibodies, have a molecular size that is below the renal clearance threshold, i.e. 60 kDa, and they are therefore rapidly eliminated through the kidneys. For addressing the issue of increasing the half-life of proteins,32 as well as that of enzymatic degradation, limited water solubility, or nonlinear pharmacokinetic profiles, several strategies have been developed. PEGylation is the well-established method, being primarily applied for


Page 16 of 39

Page 17 of 39 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


prolonging the circulation of drugs or of drug delivery systems. PEGylation was for the first time applied in 1977 by conjugating monomethoxy-PEG to bovine serum albumin,33 becoming, since that time, the golden standard for enhancing protein circulation while simultaneously being biocompatible and displaying low immunogenicity. However, PEG is not biodegradable and therefore various alternative polymeric systems are currently under scrutiny. Given that PEG exhibits high hydrophilicity and high flexibility, natural or semisynthetic polysaccharides, sharing similar properties, have been examined as substitutes including Dextran, Polysialic acid, Hyaluronic acid, Dextrin and Hydroxyethyl starch. They are all highly hydrophilic, biodegradable, non-immunogenic and non-toxic and they have been conjugated with a diversity of proteins34-36 or nanoparticles as recently reviewed.37,38 In a recent review34 a rather detailed section was devoted to these polysaccharides, in which their structural characteristics and properties to conjugate with selected proteins, was described.34 However, a direct comparison of the properties of their conjugates with proteins/peptides, which could only be obtained if all of them were interacting with the same compound, for instance with the same protein, is yet not available. It should be noted that structural differentiations among the polysaccharides impose changes in their conjugates, e.g. in their size, charge, shape, and stability. HES due to its high water solubility and its tunable hydrolysis, that is due to the presence of hydroxyethyl group at varying concentrations at its polymeric backbone, and given that it is already approved for medical use, is considered a potential substitute of PEG. Indeed the first two studies have been published also enabling a direct comparison between HES and PEG. HESylation of proteins was achieved by site-specific modification of HES before interacting with target proteins.32,39 This results in size increase, while biodegradability as well as other advantageous properties of the protein are maintained. Two examples of HES–protein conjugates, underlining the impact of this strategy on the properties of proteins, while highlighting on the extension of their half-life will be discussed below. 3.1 HESylated Anakinra Anakinra is a protein of 17.26 kDa that blocks the inflammatory action of IL-1 through binding to IL-1 receptor. It has been approved for rheumatic arthritis and recommended for patients with an insufficient response to other anti-rheumatic drugs.40,41 It has to be



Page 18 of 39

administrated at a daily dose of 100 mg, which is apparently, inconvenient and costly. HESylated Anakinra was prepared and its physicochemical properties were compared to those of the native protein. Finally, the binding affinity and pharmacokinetic properties of the modified protein were assessed. Anakinra was coupled to an activated HES derivative (Mn = 65 kDa, polydispersity = 1.3) that bears at its N-terminus an aldehydic end-group. A highly purified mono-HESylated protein was obtained with a 65% yield. The hydrodynamic size of this conjugate determined by DLS (14.7 nm) is about three times as that of native Anakinra which was found to be 4.36 nm, in agreement with values reported in the literature.40 The FTIR spectrum of HESylated Anakinra is almost identical to that of native protein in the Amide I region, suggesting that HESylation did not lead to a significant change in its secondary structure. Microcalorimetry indicates that Anakinra in water has a melting temperature of 58.0 ± 0.5 oC, in line with literature, whereas HESylated Anakinra exhibits a 4.8 oC temperature increase in the melting point, it has almost double melting enthalpy and more interestingly it retains the ability to refold upon cooling by ~90%. This is in contrast with native protein that does not exhibit any reversibility (Table 1).

Table 1. Microcalorimetry results of native and HESylated anakinra water solutions (reprinted from ref. 40).

Native anakinra

58.0 ± 0.5

∆H (kcal/mol) 58.4 ± 0.1

HESylated anakinra

62.8 ± 0.3

100.8 ± 1.0

Sample

Tm (oC)

∆cp (kcal/mol/oC) 11.7 ± 0.4

Unfolding reversibility (%) 0

19.4 ± 0.3

90.3 ± 0.9

The plasma half-life of HESylated Anakinra is significantly increased (~ 6.5 times) compared to native Anakinra. HESylated

Anakinra after single intravenous

administration to rats exhibits reduced clearance and significant increase (~ 45 times) of bioavailability, as denoted by the area under the plasma concentration curve. It becomes therefore clear that HESylation of Anakinra modifies drastically its physicochemical properties and, most importantly, leads to a half-life extension or, otherwise, increases the


Page 19 of 39 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


circulation time. Due to these properties HES was proposed as a potential substitute of PEG. In a recent study,42 HESylated versus PEGylated anakinra was compared, both prepared by employing reductive amination method. It was shown that both derivatives do not cause changes on the secondary structure of the protein, exhibit significantly reduced protein affinity and similarly increase the melting temperature in water. However, HESylated anakinra concentrated solutions had 40% lower viscosity and were superior regarding storage at 40°C. 3.2

HESylated Erythropoietin

Erythropoietin (EPO) is a glycoprotein hormone32 used extensively in the treatment of anemia due to cancer and kidney diseases. Its disadvantage is due to the fast renal clearance attributed to its relatively small size of ∼30 kDa. The introduction of two additional N-glycosylation sites was found to increase the in vivo half-life and threefold enhanced its efficacy. Alternatively, it is possible to enhance its efficacy using half-life prolongation technologies such as PEGylation or by stimulating red blood cell production using artificial activating peptide ligands for the EPO receptor. HESylation of EPO was conducted using two different conjugation methodologies. According to the first strategy, this was accomplished by targeting the glycosylation sites used for the introduction of the polymer while, according to the second strategy, by employing a reductive procedure that allows the preferential modification of protein’s Nterminal α-amino group. In order to overcome the liver clearance mechanism, the glycan moieties of EPO were selectively shielded with HES. This was achieved either by selective oxidation of sialic acid residues under mild conditions, or alternatively by employing an enzymatic method for the formation of carbonyl functionalities, which were subsequently reacted with the aldehyde end-group of activated HES. HESylated EPO is produced by Fresenius-Kabi and exhibits in vitro and in vivo activities that are comparable to those of Mircera™. The latter is the commercial PEGylated EPO (a 30 kDa PEG-EPO conjugate) which exhibits a 3-fold increase in half-life over the wild-type protein.32,39 HES-EPO conjugates showed significant residual in vitro activity employing the UT - 7 cell line and using cell viability as a measure of the EPO-dependent stimulation of proliferation. Activities of the two types of HES-EPO conjugates, i.e. HES-EPO N-



terminal and HES-EPO Glyco, were compared to native EPO, as well as to hyperglycosylated EPO (Aranesp®) and PEG-EPO (Mircera™). Both HESylated EPO derivatives exhibited (Figure 10) significant in vitro activity, ranging between 20 to 40% of the EPO control. Additionally, the effect of various modifications on the in vivo efficacy of the respective EPO compounds was tested in a simple mouse model employing the value of hematocrit after subcutaneous (s.c.) injection that was used as indicator of the erythropoietic activity. As it is shown in Figure 11, natural EPO elicits only a weak and temporary response due to its fast renal excretion, but both HESylated EPO derivatives resulted in significantly increased hematocrit values Together with the PEG-EPO (Mircera™) have the highest half-life compared to native EPO or Aranesp® that is today’s standard treatment.

Figure 10. In vitro proliferative response of UT - 7 cells after treatment with EPO and EPO - polymer conjugates was measured in a WST-1 viability assay (reprinted from ref. 32).


Page 20 of 39

Page 21 of 39 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 11. Erythropoietic in vivo activity of EPO and EPO - polymer conjugates followed by measurement of the hematocrit in BALB/C mice dosed with 50 µg kg −1 of the respective erythropoietin test substance subcutaneously (reprinted from ref. 32).

4. HES-based Nanocapsules as prospective drug delivery systems Taking into consideration the previously discussed advantageous properties of HES in delivering selected anticancer molecules or therapeutic proteins, a new strategy will be presented in this chapter in which the bioactive compounds are encapsulated inside HESbased nanocapsules. These nanocapsules should be small, preferably below 300 nm, able to be functionalized and to deliver a high concentration of bioactive molecules. Nanocapsules consisting of an aqueous core, enclosed by a HES derived polymeric coat, were prepared by an interfacial polyaddition reaction in inverse miniemulsion.43 The reaction between the hydroxyl groups of HES and the isocyanate groups of toluene diisocyanate (TDI) is taking place at water-in-oil droplet interphase giving rise to a polymeric cell. The amount of TDI employed was lower to the amount of hydroxyl groups of HES and, after redispersion of the capsules in the aqueous phase, the remaining hydroxyl groups were converted to carboxylic groups by carboxymethylation. These carboxylic groups were used for the covalent coupling of the amine-terminated folic acid achieved using EDC-mediated coupling. The amount of the attached folic acid moieties was determined from fluorescence intensity measurements. After the reaction, folate retains its receptor binding affinity due to the presence of a spacer between the folic acid and the carboxyl group on the capsule’s surface and it is therefore possible to be used



further in a receptor-mediated endocytosis. The nanocapsules obtained are stable since no precipitation or aggregation was observed within 3 months under ambient conditions. SEM studies of HES nanocapsules of an average size of 275 nm (Figure 12) confirm the formation of a core−shell structure and that the morphology did not alter upon coupling of folic acid.

Figure 12. SEM observations of HES-folic acid conjugate revealing the presence of large and small capsules (reprinted from ref. 43).

HES nanocapsules before and after folic acid coupling were evaluated for potential cytotoxicity using the MTS assay and also they were subjected to cell uptake experiments. It was found that the metabolic activity of the cells was not significantly reduced following incubation for 24 h with either redispersed HES nanocapsules (HESR) or folate-coupled HES nanocapsules (HES-FA). Additionally, very low cellular uptake was observed with HES-R nanocapsules that were not subjected to any further surface modifications. Conjugation with folic acid led at a specific cellular uptake of the HES-FA capsules into folate receptor positive HeLa cells as shown in Figure 13. The folate acid receptor (FRa)-mediated uptake in HeLa cells was confirmed primarily for HES nanocapsules of a lower size fraction (HES-FA-F, 174 nm average size). Uptake was partially inhibited when free folic acid was added in the culture medium since additional folic acid competitively inhibits FRa-mediated uptake. On the other hand control experiments with folate receptor negative A549 cells showed a very low cellular uptake for both non-conjugated and folic acid-conjugated HES nanocapsules. Overall, folate-


Page 22 of 39

Page 23 of 39 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


coupled HES based nanocapsules prove effective in targeting folate receptor positive cells.

Figure 13. Cellular uptake in HeLa cells of redispersed HES (HES-R) and folate-coupled HES (HES-FA or HES-FA-F) nanocapsules after 2 and 24 h incubation, which was performed either in the absence of folic acid (0.0 mM) or with 0.1, 0.5, and 1.0 mM folic acid in DMEM. HES-FA nanocapsules had an average size of 307 nm, HES-FA-F nanocapsules had an average size of 174 nm (reprinted from ref. 43).

Detailed information about the intracellular localization of HES-R, HES-FA, and HES-FA-F nanocapsules in HeLa and A549 cells was obtained by Confocal Laser Scanning Microscopy (CLSM). The intracellular uptake after 24 h in HeLa cells is shown in Figure 14. Higher uptake was observed for HES-FA-F than for HES-FA, and only limited uptake for HES-R. It should be noted that nanoparticle uptake did not lead to any morphological change to the HeLa cells and that it was lower when folic acid was present in the medium. On the contrary, uptake for the FR-negative A549 cells was hardly detectable.



Figure 14. Confocal Laser Scanning Microscopy images of HeLa cells showing uptake and localization of HES-R, HES-FA, and HES-FA-F nanocapsules after a 24 h incubation period in the absence (first row) or presence (second row) of folic acid (1.0 mM) in the medium. HES nanocapsules are pseudocolored in red, cell membrane in green (reprinted from ref. 43). In analogy to the previous report, hydroxyethyl starch was also utilized for the formation of nanocapsules44 in which several principles of targeted drug delivery are applied. These nanocapsules were prepared through an interfacial polyaddition reaction in inverse miniemulsion. The miniemulsification, obtained by ultrasonication, afforded nano-sized droplets that were kinetically stable. The added TDI reacted with hydroxyl groups originating from HES and yield a cross-linked polymeric shell. SEM and TEM investigations proved that the nanocapsules had a thickness ranging between 10 to 20 nm and core-shell morphology, Figure 15.


Page 24 of 39

Page 25 of 39 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 15. SEM (left) and TEM (right) images of HES-based nanocapsules (reprinted from ref. 44).

Following the synthetic process the unreacted isocyanate groups remaining on the surface were transformed to amine groups after their dispersion in water. The amine groups were used for the introduction of a PEG spacer on the surface of the nanocapsules for enhancing the accessibility of the targeting ligand. Cross-linked HES-based nanocapsules (HES NCs) were decorated with oligo(mannose) which is a targeting ligand for macrophage and dendritic cells. In this case, amine groups were reacted either directly with the isothiocyanate group of a mannose (Man) derivative, ITC-Man, or through reductive amination of mannose dimer or trimer (di-Man and tri-Man), affording HESbased nanocapsules with one, two or three mannose units, as shown in Figure 16.

Figure 16. Functionalization of HES-based nanocapsules with a-D-mannopyranosylphenyl isothiocyanate (ITC-Man), 3-O-(a-D-mannopyranosyl)-D-mannose (di-Man) and a3,a6-mannotriose (tri-Man) (reprinted from ref. 44).



The effectiveness of the mannose decorated nanocapsules to interact with the fluorescein isothiocyanate (FITC)-labeled galanthus nivalis agglutinin (GNA-FITC) is dependent on the mannose type, the density of mannose and the presence of the PEG spacer. As shown in Figure 17, the fluorescence intensity of the non-modified HES nanocapsules is always higher than that of mannose decorated nanocapsules which provide evidence that mannose can effectively bind lectin. Fluorescence intensity was also higher for mannose decorated nanocapsules bearing a PEG spacer, due to the better accessibility of the targeting ligand. A comparison of different mannose molecules introduced on the nanocapsule surface shows a stronger interaction for 3-O-(a-Dmannopyranosyl)-D-mannose (di-Man) and a3,a6-mannotriose (tri-Man) in comparison to ITC-Man decorated nanocapsule in spite of a lower density. This observation is attributed to the higher binding affinity of the lectin for these sugars, despite the fact that in the present case one sugar is in the open form.

Figure 17. Fluorescence of washed NC dispersion measured after interaction between mannosylated HES NCs and GNA-FITC: (A) non-modified HES NCs; (B) HES NCs with ITC-Man; (C) HES NCs with di-Man; (D) HES NCs with tri-Man; (E) HES NCs with a PEG linker; (F) HES NCs with PEG/ITC-Man; (G) HES NCs with PEG/di-Man; (H) HES NCs with PEG/tri-Man. Di-Man, tri-Man, PEG correspond to 3-O-(a-Dmannopyranosyl)-D-mannose, a3,a6-mannotriose and the PEG linker, respectively (reprinted from ref. 44).


Page 26 of 39

Page 27 of 39 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Dendritic cells in immunological applications have been investigated for vaccination trials and they are also targets in other immunological diseases. Nanocapsules uptake has been highly enhanced by the mannose on their surface as studied by flow cytometry. By using confocal laser scanning microscopy it has been shown that mannose functionalized nanocapsules are taken-up into the target cells, Figure 18. They are therefore promising drug carriers for targeted delivery to dendritic cells.

Figure 18. Confocal laser scanning microscopy of NCs. The cytoplasm is stained in green, nuclear membrane in blue and NCs in red. Non-modified HES NCs were nonspecifically only attached to the cells (A), whereas mannosylated HES/tri-Man NCs were taken up to a much greater extent (B) (reprinted from ref. 44).

5. HES-based Hydrogels as prospective protein delivery systems Efficient drug delivery systems are in general required for therapeutic proteins and since hydrogels show certain resemblance to natural living tissues due to their high water content and soft consistency, investigations have recently been directed towards the preparation and application of hydrogels as drug carriers for proteins.45 In addition, the high water content of hydrogels contributes to their biocompatibility and the nondestructive encapsulation of proteins. Τhe release of proteins from hydrogel networks, as an outcome of their degradation and/or diffusion, can effectively be fine-tuned optimizing in certain cases their properties. A synthetic strategy to hydrogels formation based on HES, involves the introduction of a polymerizable group at its side chain, followed by cross-linking through photopolymerization.46 As polymerizable group, hydroxyethyl methacrylate (HEMA) was



attached to the HES backbone affording HES-hydroxyethyl methacrylate (HES-HEMA), 7, following the activation with carbonyldiimidazole. In this manner hydrogels are degradable under physiological conditions.

7, HES-HEMA

The degree of substitution (DS), i.e. the amount of cross-linkable HEMA moieties on the hydroxyethyl starch backbone, determines the cross-linking density and the release properties of the hydrogels. If DS is smaller than 0.04 a gel cannot be prepared. Systems with DS higher than 0.25 are not completely water-soluble, while for hydrogels with a DS between 0.04–0.25, the swelling ratio is ranging from 600–1000%. In this case crosslinking was completed by exposing an aqueous two phase system to UV-light. For modeling the delivery of proteins with HES-derived hydrogels and for evaluating the influence of DS and Mn on the release profile, fluorescence-labeled lysozyme and dextran of various molecular weights (20 kDa, 70 kDa and 500 kDa) were encapsulated. The release studies were carried out at pH 7 and at 37 °C. Τhe formation of microparticles was achieved by using a water-in-water (w/w) emulsion process, Figure 19. The HES-HEMA and the selected protein were dissolved in water and mixed with an aqueous solution of polyethylene glycol. The two polymeric solutions are not miscible and an emulsion was formed. Subsequently the HES-HEMA phase is photopolymerized. The process was optimized for obtaining a narrow distribution of particle sizes. Thus, the ratio of the HES-HEMA and PEG solutions, the temperature, the mixing procedure and the amount of protein were varied and the size and size-distribution of particles was determined.


Page 28 of 39

Page 29 of 39 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 19. Schematic representation of the manufacturing method for HES–HEMA microparticles (reprinted from ref. 46).

The so-produced spherical microparticles show a monomodal particle size distribution with an average size of about 10 µm.47 It is evident that by increasing the network density the release rate is decreased. It was found that small molecules are retained at a lower extent than molecules of high molecular weight (Figure 20). The results of the initial release rate suggest that hydrogels with DS 0.05 and 0.22 differ primarily on the number of pores at approximately 6.6 nm corresponding to ~70 kDa (Figure 21). Molecules which exceed this size were only released after degradation of the hydrogel, which was very slow in PBS at pH 7. For accelerating biodegradation of the hydrogel and therefore the release of FITC-dextran, loaded microspheres were incubated in sodium carbonate buffer at pH 9.6 and human serum, respectively. In sodium carbonate the hydrolytically labile carbonate group of HES-HEMA is rapidly degraded (Figure 22A). Incubation in human serum degrades the hydrogel network due to the presence of α-amylase. This is able to degrade the HES-backbone at positions 1–4, therefore accelerating the release (Figure 22B).



Figure 20. Release of FITC-dextran of various molecular weights (20 kDa ●○; 70 kDa □■; 500 kDa ∆▲) from microspheres with degree of substitution (DS) 0.05 (open symbols) or 0.22 (filled symbols) (reprinted from ref. 47).

Figure 21. Effect of the degree of substitution (DS) of HES-HEMA on the release of fluorescence-labeled lysozyme from microspheres. (DS=0.05 ○; DS=0.07 ▲; DS=0.14 ■; DS=0.17 □) (reprinted from ref. 47).


Page 30 of 39

Page 31 of 39 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 22. (a) Release of FITC-dextran of various molecular weights from microspheres with DS 0.05 and 0.22 at pH 9.6 (b) Release of FITC-dextran (70 kDa) from microspheres with DS 0.14 in human serum in comparison to sodium phosphate buffer (reprinted from ref. 47).

The described method is therefore promising for the preparation of drug loaded hydrogel microspheres. The release of FITC-dextran is bi-phasic, exhibiting a rapid release of predominantly small molecules by diffusion and a slow release after hydrolytic degradation of the hydrogel. The degree of substitution of the polymer affects both phases, which allows tuning the release profile of HES-HEMA derived hydrogels.



Page 32 of 39

6. Concluding Remarks and Outlook Functional drug delivery systems have been conveniently prepared by interacting HES, or slightly modified HES derivatives, with bioactive molecules or macromolecules including proteins. Depending on the nature of the interacting compounds covalently conjugated or non-covalently bonded systems were obtained. The majority of todays investigated conjugates were covalently linked. In the present topical review, well-known anticancer compounds and therapeutic proteins were used for the preparation of the corresponding HESylated conjugates. Following the conjugation stage, drug formulations were obtained that were water soluble, biocompatible, biodegradable, had low toxicity and most importantly their half-life was extended in plasma as discussed in the examples above. Additionally, nanocapsules and hydrogels based on HES were prepared under facile synthetic conditions, as models for prospective drug delivery systems. The published results are encouraging, although HES utility as a novel DDS has to be further verified with more studies, which we hope to trigger with this topical review. Furthermore, it is envisaged that in the near future molecular engineering applied on the above systems by introducing targeting ligands, molecular transporting moieties or fluorescence probes

will

lead

to the preparation

of multifunctional

and/or

multicompartment drug delivery HES-based systems with improved specificity and efficacy. Research activity on the development of similar systems is continuing with a fast pace as it is clearly demonstrated by several excellent reviews and perspectives48-53 dealing with drug delivery systems which are analogous to the present review.

ABBREVIATIONS HES, hydroxyethyl starch; DDSs, Drug Delivery Systems; PEG, polyethylene glycol; Mn, number average molecular weight; Ms, degree of HES substitution with hydroxyethyl groups; C-HES, carboxylated hydroxyethyl starch; DOX, doxorubicin; CLSM, confocal laser scanning microscopy; DIC, differential interference contrast; DAPI, 4',6-diamidino2-phenylindole;

MTT,

3-4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium

bromide;

GSH, glutathione; 10-HCPT, 10-hydroxy camptothecin; PBS, phosphate-buffered saline; 5-FU, 5-fluorouracil; FUAC, 5-fluorouracil-1-acetic acid; MTX, methotrexate; EPR,


Page 33 of 39 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


enhanced permeability and retention effect; NOD, non-obese diabetic; SCID, severe combined immunodeficiency; DLS, dynamic light scattering; FTIR, Fourier transform Infrared spectroscopy; EPO, erythropoietin; TDI, toluene diisocyanate; SEM, scanning electron microscopy; TEM, transmission electron microscopy; NC, nanocapsule; ITC, isothiocyanate; Man, mannose; GNA, galanthus nivalis agglutinin; FTIC, Fluorescein isothiocyanate; HEMA, hydroxyethyl methacrylate; DS, degree of substitution; w/w, water-in-water emulsion.

REFERENCES (1) Bangham, A. D., Standish, M. M. and Watkins, J. C. (1965) Diffusion of univalent ions across lamellae of swollen phospholipids. J. Mol. Biol. 13, 238–252. (2) Ringsdorf, H. (1975) Structure and properties of pharmacologically active polymers. J. Polym. Sci., C, Polym. Symp. 51, 135–153. (3) Liu, Z., Jiao, Y., Wang, Y., Zhou, C., and Zhang Z. (2008) Polysaccharides-based nanoparticles as drug delivery systems. Adv. Drug Delivery Rev. 60, 1650-1662. (4) Zhang, N., Wardwell, P. R., and Bader, R. A. (2013) Polysaccharide-based micelles for drug delivery. Pharmaceutics 5, 329-352. (5) Marchessault, R. H., Ravenelle, F., and Zhu, X. X. (2006) Polysaccharides for Drug Delivery and Pharmaceutical Applications. ACS Symposium Series. Volume 934, pp 57, Chapter 3, American Chemical Society, Washington, DC, USA. (6) Debele, T. A., Mekuria, S. L., and Tsai, H.-C. (2016) Polysaccharide based nanogels in the drug delivery system: Application as the carrier of pharmaceutical agents. Mat. Sci. Eng. C 68, 964–981. (7) Boldt, J. (2009) Modern degradable hydroxyethyl starches: current concepts. Anesth. Analg. 108, 1574-1582. (8) Besheer, A., Hause, G., Kressler, J., and Mäder, K. (2007) Hydrophobically modified hydroxyethyl starch: Synthesis, characterization, and aqueous self-assembly into nano-sized polymeric micelles and vesicles. Biomacromolecules 8, 359-367.



(9) Narayanan, D., Nair S., and Menon, D. (2015) A systematic evaluation of hydroxyethyl starch as a potential nanocarrier for parenteral drug delivery. Int. J. Biol. Macromolecules 74, 575-584. (10) Dellacherie, E. (1996) Polysaccharides in medical applications. pp 525-544, Marcel Dekker, Inc., New York, USA. (11) Sibylle, A., and Kozek-Langenecker, M. D. (2005) Effects of hydroxyethyl starch solutions on hemostasis. Anesthesiology 103, 654–660. (12) Noga, M., Edinger, D., Kläger, R., Wegner, V. S., Spatz, J. P., Wagner, E., Winter, G., and Besheer, A. (2013) The effect of molar mass and degree of hydroxymethylation on the controlled shielding and deshielding of hydroxyethyl starchcoated polyplexes. Biomaterials 34, 2530-2538. (13) Schortgen, F., Deye, N., and Brochard, L. (2004) Preferred plasma volume expanders for critically ill patients: results of an international survey. Intensive Care Med. 30, 2222-2229. (14) Zarychanski, R., Abou-Setta, A. M., Turgeon, A. F., Houston, B. L., McIntyre, L., Marshall, J. C and Fergusson, D. A. (2013) Association of hydroxyethyl starch administration with mortality and acute kidney injury in critically ill patients requiring volume resuscitation. A systematic review and meta–analysis. JAMA 309, 678-688. (15) Paleos, C. M., Sideratou, Z., Theodossiou, T. A., and Tsiourvas, D. (2015) Carboxylated hydroxyethyl starch: A novel Polysaccharide for the delivery of doxorubicin. Chem. Biol. Drug Des. 85, 653-658. (16) Mohan, P., and Rapoport, N. (2010) Doxorubicin as a molecular nanotheranostic agent: effect of doxorubicin encapsulation in micelles or nanoemulsions on the ultrasound-mediated intracellular delivery and nuclear trafficking. Mol. Pharm. 7, 1959– 1973. (17) Paleos, C. M., Tsiourvas, D., Sideratou, Z., and Tziveleka, L.-A. (2010) Drug delivery employing multifunctional dendrimers and hyperbranched polymers. Expert Opin. Drug Del. 7, 1387-1398.


Page 34 of 39

Page 35 of 39 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(18) Laginha, K. M., Venwoert, S., Charrois, G. J., and Allen, T. M. (2005) Determination of doxorubicin levels in whole tumor and tumor nuclei in murine breast cancer tumors. Clin. Cancer Res. 11, 6944-6949. (19) Zhu, Y., Yao, X., Chen, X., and Chen, L. (2015) pH-sensitive hydroxyethyl starch-doxorubicin conjugates as antitumor prodrugs with enhanced anticancer efficacy. J. Applied Polym. Sci. 132, DOI:10.1002/App.42778. (20) Hu, H., Li, Y., Zhou, Q., Ao, Y., Yu, C., Wan, Y., Xu, H., Li, Z., and Yang, X. (2016) Redox-sensitive hydroxyethyl starch-doxorubicin conjugate for tumor targeted drug delivery. ACS Appl. Mat. Interfaces 8, 30833-30844. (21) Li, G., Li, Y., Tang, Y., Zhang, Y.; Zhang, Y., Yin, T., Xu, H., Cai, C., and Tang, X. (2014) Hydroxyethyl starch conjugates for improving the stability, pharmacokinetic behavior and antitumor activity of 10-hydrroxy camptothecin. Int. J. Pharm. 471, 234244. (22) Zhang, L., Yang, M., Wang, Q., Li, Y., R. Guo, R, Jiang, X., Yang, C., and Liu, B. (2007) 10-Hydroxycamptothecin loaded nanoparticles: Preparation and antitumor activity in mice. J. Controlled Release 119, 153-162. (23) Brockman, R., and Anderson, E. (1963). Biochemistry of cancer (metabolic aspects). Annu. Rev. Biochem. 32, 463–512. (24) Luo, Q., Wang, P., Miao, Y., He, H., and Tang, X. (2012) A novel 5-fluorouracil prodrug using hydroxyethyl starch as a macromolecular carrier for sustained release. Carbohyd. Polym. 87, 2642-4647. (25) Goszcyński, T. M., Filip-Psurska, B., Kempińska, K., Wietrzyk, J., and Boratyński, (2014). Hydroxyethyl starch as an effective methotrexate carrier in anticancer therapy. Pharma. Res. Per. 2, DOI: 10.1002/prp2.47. (26) Nevozhay, D., Budzynska, R., Kanska, U., Jagiello, M., Omar, M. S., Boratynski, J., and Opolski, A. (2006) Antitumor properties and toxicity of dextran-methotrexate conjugates are dependent on the molecular weight of the carrier. Anticancer Res. 26, 1135–1143.



(27) Torchilin, V. (2011) Tumor delivery of macromolecular drugs based on the EPR effect. Adv. Drug Delivery Rev. 63, 131–135. (28) Pisal, D. S., Kosloski, M. P., and Balu-lyer, S. V. (2010) Delivery of therapeutic proteins. J. Pharm. Sci. 99, 2557-2575. (29) Rehman, K., Hamid Akash, M. S., Akhtar, B., Tariq, M., Muhmood, A., and Ibrahim M. (2016) Delivery of therapeutic proteins: Challenges and Strategies. Curr. Drug Targets 17, 1172-1188. (30) Besheer, A., Liebner, R., Meyer, M. and Winter, G. (2013) Challenges for PEGylated proteins and alternative half-life extension technologies based on biodegradable polymers. ACS symposium series, chapter 13, ACS Washington Washington DC, USA. (31) Kontermann, R. E. (2011) Strategies for extended serum half-life of protein therapeutics. Curr. Opin. Biotechnol. 22, 868-876. (32) Hey, T., Knoller, H., and Vorstheim, P. (2012) Therapeutic Proteins: Strategies to modulate their plasma half-lives. Wiley-VCH, Weinheim, Germany. (33) Abuchowski, A., van Es, T., Palczuk, N. C., and Davis, F. F. (1977) Alteration of immunologiocal properties of bovine serum albumin by covalent attachment of polyethylene glycol. J. Biol. Chem. 252, 3578-3581. 219-233. (34) Pasut, G., (2014), Polymers for Protein Conjugation, Polymers 6, 160-178. (35) Styslinger, T. J., Zhang, N., Bhatt, V. S., Pettit, N., Palmer, A. F., and Wang, P. G. (2012) Site-selective glycosylation of Hemoglobin with variable molecular weight oligosaccharides: potential alternative to PEGylation. J. Am. Chem. Soc. 134, 7507-7515. (36) Gregoriadis, G., Jain, S., Papaioannou, I. and Laing, P. (2005) Improving the therapeutic efficiency of peptides and proteins: A role for polysialic acids. Int. J. Pharm. 300, 125-130. (37) Salmaso, S., and Caliceti, P. (2013) Stealth Properties to Improve Therapeutic Efficacy of Drug Nanocarriers. J. Drug Delivery, DOI.org/10.1155/2013/374252.


Page 36 of 39

Page 37 of 39 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(38) Amoozgar, Z., and Yeo, Y. (2012) Recent advances in stealth coating of nanoparticle drug delivery systems. WIREs Nanomed Nanobiotechnol 4, 219-233. (39) Pelegri-O’Day, E. M., Lin, E.-W., and Maynard, H. D. (2014) Therapeutic protein –polymer conjugates: Advancing beyond PEGylation. J. Am. Chem. Soc. 136, 14323-14332. (40) Liebner, R., Mathaes, R., Meyer, M., Hey, T., Winter, G., and Besheer, A. (2014) Protein

HESylation

for

half-life

extension:

synthesis,

characterization

and

pharmacokinetics of HESylated anakinra. Eur. J. Pharm. Biopharm. 87, 378-385. (41) Burger, D. and Dayer, J. M. (2012) IL-1Ra, Cytokine Reference (Oppenheim, J. J. and Feldmann, M, Eds.) pp. 319–336, Academic Press, London. (42) Liebner, R., Meyer, M., Hey, T., Winter, G., and Besheer, A. (2015) Head to head comparison of the formulation and stability of concentrated solutions of HESylated versus PEGylated anakinra. J. Pharm. Sci. 104, 515–526. (43) Baier, G., Baumann, D., Siebert, J. M., Musyanovych, A. Mailänder, V., and Landfester, K. (2012) Suppressing unspecific cell uptake for targeted delivery using hydroxyethyl starch nanocapsules, Biomacromolecules 13, 2704-2715. (44) Freichels, H., Wagner, M., Okwieka, P., Meyer, R. G., Mailänder, V., Landfester, K., and Musyanovych, A. (2013) (Oligo)mannose functionalized hydroxyethyl starch nanocapsules: en route to drug delivery systems with targeting properties. J. Mater. Chem. B 1, 4338-4348. (45) Schwoerer, A. D. A., Harling, S., Scheibe, K., Menzel, H., and Daniels, R. (2009) Influence of degree of substitution of HES-HEMA on the release of incorporated drug models from corresponding hydrogels. Eur. J. Pharm. Biopharm. 73, 351-356. (46) Harling, S., Schwoerer, A., Scheibe, K., Daniels, R., and Menzel, H. (2010) A new hydrogel drug delivery system based on hydroxyethyl starch derivatives. J Microencapsul. 27, 400-408. (47) Schwoerer, A., Harling, S., Menzel, H., and Daniels, R. (2008), Release behavior of hydrogel microparticles based on hydroxyl-ethyl-starch as a drug delivery system for proteins. J. Controlled Release 132, e16-e17.



(48) Stanzl, E. G., Trantow, B. M., Vargas, J. R., and Wender, P. A. (2013) Fifteen years of cell-penetrating, guanidinium-rich molecular transporters: basic science, research tools, and clinical applications. Acc. Chem. Res. 46, 2944-2954. (49) Coburn, J. M., and Kaplan, D. L. (2015) Engineering Biomaterial-Drug Conjugates for local and sustained chemotherapeutic delivery. Bioconjugate Chem. 26, 1212–1223. (50) Buwalda, S. J., Vermonden, T., and Hennink, W. E. (2017) Hydrogels for therapeutic delivery: Current developments and future directions. Biomacromolecules DOI: 10.1021/acs.biomac.6b01604. (51) Paleos, C. M., Tsiourvas, D., and Sideratou, Z. (2016) Triphenylphosphonium Decorated Liposomes and Dendritic Polymers: Prospective Second Generation Drug Delivery Systems for Targeting Mitochondria. Mol. Pharm. 13, 2233–2241. (52) Maity, A. R., and Stepensky, D. (2016) Limited Efficiency of Drug Delivery to Specific Intracellular Organelles Using Subcellularly “Targeted” Drug Delivery Systems. Mol. Pharmaceutics 13, 1-7. (53) Sharma, A. R., Kundu, S. K., Nam, J.-S., Sharma, G., Doss, C. G. P., Lee, S.-S. and Chakraborty, C. (2014) Next Generation Delivery System for Proteins and Genes of Therapeutic Purpose: Why and How? BioMed Res. Inter. DOI:10.1155/2014/327950.


Page 38 of 39

Page 39 of 39 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Table of contents graphic


Drug Delivery Systems Based on Hydroxyethyl Starch - American

Recommend Documents