Nanopharmaceutical formulations based on poly ...

Bulgarian Journal ofChemistry Volume 3 Issue 1 Article history: Received: 27 March 2014 Revised: 26 April 2014 Accepted: 27 April 2014 Available online: 12 May 2014

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Nanopharmaceutical formulations based on poly(styrene-co-maleic acid) Nadezhda Angelova, Georgi Yordanov* Faculty ofChemistry and Pharmacy, Sofia University “St. Kliment Ohridski”, 1 James Bourchier Blvd., 1164 Sofia, Bulgaria *Corresponding author. E-mail: [email protected]

Copolymers of styrene and maleic acid of relatively low molecular weight possess a great potential for application as components of drug delivery systems, especially in cancer chemotherapy. The pioneering research in this area has been performed in the 1980s during the development ofmacromolecular complexes for selective drug delivery to solid tumors. Currently, there are some quite promising formulations of anti-cancer agents, based on these copolymers, that have shown a remarkable antitumor activity when tested in preclinical and clinical trials. The aim of the current review is to summarize the basic approaches for the preparation of micellar and nanoparticle formulations based on poly(styrene- co -maleic acid) (PSMA), the main strategy to deliver such systems to solid tumors, their toxicity, pharmacokinetics and efficacy in preclinical and clinical trials. The current status and future perspectives of using these delivery systems are discussed, including recent contributions of the authors about preparation ofPSMA nanoparticles by nanoprecipitation approach. Ключови думи: Drug delivery, EPR effect, micelles, nanoparticles, poly(styrene-co-maleic acid), solid tumors.

Съдържание:

Nadezhda Angelova is studying Computational Chemistry in Sofia University and she has done one exchange semester in the University ofBarcelona as Erasmus student. She is interested in biomedical and pharmaceutical nanotechnology and since 2013 is working under the guidance ofDr. Georgi Yordanov in researching nanopharmaceutical formulations as drug delivery systems. Dr. Georgi Yordanov is a lecturer in biomedical and pharmaceutical nanotechnology at the Faculty ofChemistry and Pharmacy at Sofia University “St. Kliment Ohridski”. His current scientific interests are in the area ofcolloidal drug delivery systems for cancer chemotherapy. For his achievements in this area, Dr. Yordanov has received the award for best young scientist in polymer science “Prof. Ivan Shopov” (Union ofChemists in Bulgaria and the Institute of Polymers, BAS) for the year 2010, and the award for best young scientist ofSofia University in 2013.

1. Nanopharmaceuticals and nanomedicine: an introduction........................34 2. Poly(styrene-co-maleic acid): synthesis and properties..........................34 2.1. Synthesis...................................................................................................34 2.2. Physicochemical properties)...................................................................35 3. Nanocolloidal drug delivery systems ofpoly(styrene-co-maleic acid).......35 www.bjc.chimexpert.com

© 2014, SDCB Foundation, Sofia

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Nanopharmaceutical formulations based on poly(styrene-co-maleic acid)

3.1. PSMA-drug conjugates.......................................35 3.2. PSMA-based micelles ........................................35 3.3. Nanoparticles prepared by nanoprecipitation.36 4. PSMA-based nanocarriers for cancer treatment......37 4.1. Accumulation of PSMA-based nanocarriers within solid tumors..........................................................37 4.2. Intratumoral vascular damage caused by PSMAbased nanocarriers.....................................................38 4.3. Efficacy of PSMA-based formulations in cancer chemotherapy.............................................................38 5. Interaction with the immune system..........................39 6. Toxicity........................................................................40 7. Conclusions..................................................................41

1. Nanopharmaceuticals and nanomedicine: introduction

The development of new drugs and formulations is mainly motivated by the limited efficacy and/or undesirable side effects of the current ones. One possibility to modify the therapeutic index and the pharmacokinetics of a drug (its bioavailability, clearance, plasma half-life, etc.) is to make a change in its molecular structure; however, such a change is actually equivalent to synthesis of a new chemical compound and is therefore quite possible to alter the mechanism of drug action. Another possibility to modify the drug pharmacokinetics (and hopefully, the therapeutic efficacy) without changing the drug molecular structure is to prepare a new formulation of the drug. Various colloidal systems, such as polymeric micelles, various nanoparticles, nanoemulsions, liposomes, and others, have been demonstrated as useful carriers for a variety of drugs [1-3]. Such nanocolloidal systems, usually of sizes below 300 nm, have been used for parenteral drug carrier systems with the aim to improve the drug pharmacokinetics and the therapeutic efficacy, especially in cancer chemotherapy. Such formulations are generally called nanopharmaceuticals and their development and application is assigned as a part of nanomedicine, which considers the application of nanotechnology in medicine [4]. The main idea of using nanocarriers is to achieve effective delivery of a drug to the location of its action in the body thus minimizing its effective dose, side effects and increasing its therapeutic efficacy [5-7]. It is most important for systemically administered drugs, whose molecules are rapidly, and usually non-specifically, 34 Bulg. J. Chem. 3 ( 2014) 33-43

distributed throughout the body via the blood circulation after parenteral administration. The nanocarrier itself is however not simply an inert transport vehicle for the drug molecules, but could interact actively with biomolecules and living cells thus largely affecting the therapeutic outcome [8-10]. There are some general requirements for a nanocarrier system, which include submicron size (even less than 200-300 nm), good colloidal stability in body fluids, biodegradability or at least a possibility for the carrier material to be eliminated from the body after successful delivery ofthe drug cargo to its target location. Nanocarriers must not cause systemic or local organ toxicity, embolization of normal vasculature, acute immune response and other undesirable side effects. Different synthetic and natural polymers have been found to be suitable for preparation of nanocarriers – poly(lactic acid) [11], poly(alkyl cyanoacrylate) [12], poly(ε-caprolactone) [13], albumin [14], gelatine [15], chitosan [16], etc. Each of these carrier systems has advantages and disadvantages, which must be taken into account when designing nanopharmaceutical formulations. In this review we describe the current status and challenges of using poly(styrene-co-maleic acid), PSMA, as a component of nanocarrier systems for drug delivery. This article is not intended to be a complete review of this research area but just to bring together the basic concepts and ideas related to the preparation and applications of PSMA copolymers, emphasizing on their utilization in anticancer formulations. It also includes basic ideas of our new finding related to the preparation of PSMA-based nanoparticles by nanoprecipitation approach. Finally, we outline some of the current problems and the future perspectives of using PSMA copolymers in cancer chemotherapy. 2. Poly(styrene-co-maleic acid): synthesis and properties 2.1. Synthesis

The PSMA copolymers are usually synthesized by radical polymerization of a mixture of styrene and maleic anhydride, followed by hydrolysis ofthe anhydride bonds in the obtained copolymer ( Fig. 1 ). The process is classically performed in boiling (152-153 ºC) cumene by using dicumyl peroxide as a radical initiator [17,18]. The obtained copolymer anhydride is thus cumeneterminated. Copolymers of various molecular mass and


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N. Angelova анд G. Yordanov

composition (styrene/maleic anhydride ratio) are commercially available. The hydrolysis of the anhydride bonds is performed by boiling with sodium hydroxide solution for several hours, followed by acidification with hydrochloric acid yielding the PSMA copolymer. Other derivatives can be prepared from the anhydride copolymer by partial hydrolysis, reactions with alcohols to yield partial esters, etc. [18] ( Fig. 2). The anhydride bonds can also react with amines leading to formation of amide bond between the polymer and amine-containing substances (see section 3.1).

Figure 2. Preparation ofPSMA derivatives (half-esters and amide conjugates).

3. Nanocolloidal drug delivery systems of poly(styrene-co-maleic acid) 3.1. PSMA-drug conjugates

2.2. Physicochemical properties

The physicochemical properties ofPSMA copolymers are determined mainly by three factors: i) the molecular mass distribution; ii) the molar ratio of styrene to maleic www.bjc.chimexpert.com

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Figure 1. Synthesis ofpoly(styrene-co-maleic acid) (PSMA) copolymers from styrene and maleic anhydride.

acid monomer units; and iii) the relative order of monomer units in the polymer molecule (c.a. whether it is a random, alternating or block copolymer). There are also chiral centres in the copolymer molecules and therefore a possibility for various tacticity of the molecules. The PSMA copolymers are composed of hydrophobic (styrene) and hydrophilic (maleic acid) monomer units, which largely determine their amphiphilic nature [19-21]. The low molecular weight PSMA copolymers are known as excellent emulsifiers and could be used as colloidal-dispersing agents for solubilization of water-insoluble organic molecules [22]. The dispersing properties of such copolymers have been utilized also in pharmaceutical technology to prepare micellar drug formulations (see section 3.2). The adjacent carboxyl groups in the hydrophilic maleic acid monomer combined with the hydrophobic styrene in the case ofalternating copolymers may provide the flexibility for the formation of a comb-like conformation, which may explain their dispersive properties [19,22]. The alternating copolymers with equi-molar ratio of styrene to maleic acid monomer units are usually soluble in water at pH > 5 [18,23]. At acidic pH carboxylates become protonated, which decreases their water solubility. In such a way, the PSMA copolymers can be utilized for preparation of pHsensitive nanocolloids, which is highly important for pharmaceutical applications. For example, pH-sensitive liposomes have been prepared by modification with PSMA copolymer [24]. PSMA copolymers with higher content of styrene (~ 75 wt%) appear to be more hydrophobic, highly soluble in acetone (and other nonpolar organic solvents), but relatively insoluble in water at neutral pH. Our experiments with such a copolymer (Mw ~ 1900) showed that it dissolves in water at pH > 9 and is relatively insoluble at lower pH. We found that the colloidal stability of nanoparticles composed of such a copolymer is also pH-dependent and nanoparticles form aggregates at pH < 4.5 (see section 3.3).

Various water-soluble polymer-conjugated drugs have been demonstrated to possess improved pharmacokinetics in comparison with low-molecular weight drugs, such as prolonged half-life in blood,


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improved stability of the drug molecule, decreased sideeffects, etc. [25]. The first PSMA-drug conjugate has been reported by Maeda et al. in 1979 [18,26]. The conjugate (abbreviated as SMANCS) contained on average about two molecules of PSMA (1:1 halfbutylated copolymer; Mw ~ 2000) and one molecule of neocarzinostatin (an anti-cancer protein) [18]. The conjugation has been performed via amide linkage obtained after reaction between amino-groups from the protein and non-hydrolyzed anhydride groups from the copolymer. The pharmacokinetic characteristics of SMANCS have been found to be considerably different from that of neocarzinostatin: the biological half-life in blood has been prolonged 10 times, the antitumor activity had improved, and toxicity had been reduced to one-fourth [18]. SMANCS has been approved in 1994 in Japan for use in hepatoma treatment [27] and formulated with the lipid contrast medium Lipiodol (SMANCS/Lip) [28]. Many articles by Maeda et al. [18,27-31] have reported remarkable antitumor efficacy of the SMANCS formulations in clinical treatments of hepatoma, lung, renal and other cancers in humans. The SMANCS/Lip formulation has been applied via intraarterial administration (in the tumor-feeding artery) and combined with aqueous SMANCS given intravenously [28]. The improved anticancer efficacy of SMANCS has been associated with its improved accumulation in solid tumors via the EPR-effect (see section 4.1). 3.2. PSMA-based micelles

The PSMA-based micellar formulations have been prepared as carriers for anthracyclines [32,33], tanespimycin [34], zinc protoporphyrin [35,36], etc. Since anthracyclines are cationic drugs (due to protonation of an amino-group at physiological pH) they tend to interact electrostatically with the polyanionic PSMA. Hydrogen bonding between the two substances also seems possible. Taking into account that both PSMA and anthracyclines have aromatic ring structures in their molecules, and the fact that the natural fluorescence of anthracyclines is quenched upon interaction with PSMA [32,33], it is reasonable to suggest the existence of a kind of π-stacking interaction between PSMA and anthracycline molecules. PSMA-based micellar formulations of anthracyclines have been prepared by association of the anthracycline with PSMA at slightly acidic pH (~ 5) mediated by watersoluble carbodiimide (EDC; 1-Ethyl-3-(336 Bulg. J. Chem. 3 ( 2014) 33-43

Figure 3. Flowchart illustrating the preparation ofPSMA-based micelles (according to descriptions in ref. [34]).

dimethylaminopropyl)carbodiimide), followed by alkalization of the medium and reconstitution of the micelles by controlled acidification of the alkaline solution [32,33]. Schematically, the process is shown in Fig. 3 . The initial intent of using carbodiimide was to prepare an amide conjugate ofthe drug with the polymer, but structural analyses of the product indicated that no covalent conjugate was formed [32]. However, the presence ofthe EDC carbodiimide (or other amines) was necessary for the successful formation of drug-loaded micelles and therefore it has been suggested that this compound acted like a catalyst for the micelle formation. Although Maeda defined the PSMA-drug associates as “micelles”, these were actually nanoparticles (probably, of core/shell structure) of various sizes: 180 nm for PSMApirarubicin [37], 74 nm for PSMA-tanespimycin [34], 176 nm for PSMA-zinc protoporphyrin [35]. Although the sizes of these nanostructures have been well characterized, no methods for control and varying these sizes have been reported so far. 3.3. Nanoparticles prepared by nanoprecipitation

Nanoprecipitation is a classical approach for preparation of polymer colloids [38-40]. It consists of two steps ( Fig. 4): 1) preparation of polymer solution in water-miscible organic solvent; 2) mixing of this solution with aqueous medium, in which the polymer is relatively insoluble and under proper conditions (suitable polymer concentration in the organic solvent, and proper volume ratio of water to organic solvent) precipitates into nanoparticles. The organic solvent (acetone, alcohol, THF) is usually much more volatile than water and could be removed by evaporation under vacuum. In our experiments we used a PSMA copolymer (prepared by




Figure 4. Flowchart illustrating the preparation ofPSMA-based nanoparticles by nanoprecipitation in our experiments (according to descriptions in ref. [41]).


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hydrolysis of poly(styrene- co -maleic anhydride) of Mw ~1900, containing 75 wt% styrene) to prepare nanoparticles by the nanoprecipitation approach, using acetone as organic solvent and phosphate-buffered (pH 7.4) saline as aqueous medium (details about the procedure and the effects of various experimental variables on the properties of the obtained nanoparticles are given elsewhere [41]). We found that the nanoparticle size could be reproducibly controlled by varying the concentration of polymer in the acetone phase, as well as by varying the acetone/water volume ratio (Figs. 5 and 6). In such a way, nanoparticles of average sizes from 100 to 350 nm could be reproducibly obtained; larger particles were formed at higher polymer concentration and lower acetone/water ratio and vice versa. We have previously utilized a similar nanoprecipitation approach for preparation of pure and drug-loaded poly(butyl cyanoacrylate) (PBCA) nanoparticles [42-44]. Colloidal stabilizers are usually added to the aqueous medium to stabilize the nanoparticles, especially in the case of hydrophobic polymers (such as PBCA). In the case of PSMA, such stabilizers were not needed (although could be added), because nanoparticles in this case were electrostatically stabilized as a result of ionized carboxylic groups on their surface, which could be related to the observed negative zeta-potentials (from - 20 to - 30 mV) at physiologicallike conditions (PBS buffer at pH 7.4, 37 ºC). The method of nanoprecipitation could be applied only for PSMA that is relatively insoluble in aqueous media at pH ~ 7.4. For that treason we utilized PSMA enriched in styrene (75 wt%). The more common PSMA copolymers with monomer ratio 1:1 (~ 50 wt% styrene) are usually soluble in water at this pH and therefore nanoparticles can not be obtained. Drugs, such as anthracyclines, can be simply loaded by sorption to the preformed PSMA nanoparticles. For example, in our experiments epirubicin could be associated with the PSMA nanoparticles with more than 80% loading efficiency and the loaded drug was relatively strong associated with the particles. This

Figure 5. SEM images ofPSMA nanoparticles prepared by nanoprecipitation at two different acetone/water volume ratios (the concentration ofPSMA in final aqueous dispersion was 3 mg/ml): a) 1/5, b) 2/5.

Figure 6. Size distributions ofthe PSMA nanoparticles in Figure 4 obtained by DLS analysis.

approach can therefore be useful for the entrapment of highly sensitive and/or reactive drugs, which may undergo chemical transformation in the acidic/alkaline medium used for preparation of PSMA-based micelles (see section 3.2). In our experiments, we found that both PSMA and epirubicin-loaded PSMA nanoparticles were pH-sensitive and agglomerated at pH < 4.5. The agglomeration was recognized as formation of gel-like precipitates in acidic medium, which was reversible upon raising pH to neutral and ultrasonication. 4. PSMA-based nanocarriers for cancer treatment 4.1. Accumulation ofPSMA-based nanocarriers within solid tumors

Various cytostatics (neocarzinostatin [18], doxorubicin [32], pirarubicin [33], tanespimycin [34], zinc protoporphyrin IX [35,36]) have been associated


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with PSMA copolymers in order to obtain delivery systems for targeted cancer treatment. The accumulation ofPSMA-based micellar nanocarriers within solid tumors has largely been investigated by Maeda et al. [27-33]. It has been supposed that the so-called enhanced permeability and retention (EPR) effect plays a major role for achieving effective accumulation of macromolecular drugs and nanocarriers into solid tumors [31,45]. This effect is based on the pathologic anatomical structure of blood vessels and the lack of lymphatic drainage in most solid tumors. Tumor blood vessels are often fenestrated, containing openings in their walls of sizes up to 600 nm, which make it possible for small nanocarrier structures (usually less than 200 nm in size) to pass through these fenestrations and enter into the tumor interstitial space [46]. The lack of lymphatic drainage in most solid tumors is considered as the main reason for the intratumoral accumulation of nanocarriers after their passage through the fenestrations in tumor blood vessels. For achieving effective accumulation in tumor interstitium via the EPR effect nanocarriers are expected to have relatively long half-life in blood circulation (c.a., at least 6 hours). The accumulation of nanocarriers could be enhanced by increasing the permeability of blood vessels in hypoxic regions (such as tumors) after administration of nitro-glycerine [47]. Longer blood circulation of nanocarriers can be achieved by hydrophilization of their surface (for example, by modification with poly(ethylene glycol)), which minimizes adsorption of fibrinogen, immunoglobulins, complement factors and other opsonins from blood plasma onto the surface of the nanocarrier. Nanocarriers that do not adsorb opsonins from blood plasma are less recognizable by the phagocytic cells of the immune system and are therefore phagocytozed to a lesser degree, which increases their circulation lifetime [48]. Besides the need for prolonged lifetime in blood circulation, there are also other problems with exploring the EPR effect for achieving high accumulation of nanocarriers into solid tumors. Such problems include the anatomical heterogeneity of the tumor vessels and the existence of necrotic areas and thrombi inside larger tumors [31]. It should be kept in mind that the EPR effect can be explored only in vivo. Therefore, it is quite possible that conventional cytotoxicity evaluations (such as MTT assay) can show that a particular nanocolloidal drug formulation is less toxic against cancer cells in vitro than the free drug, however in vivo tests may show increased tumor accumulation (and possibly, an improved antitumor efficacy) due to the EPR effect. For example,

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such has been found to be the case of doxorubicin-loaded PSMA micelles [32]. 4.2. Intratumoral vascular damage caused by PSMA-based nanocarriers

Investigations of the effects of PSMA-pirarubicin micellar nanocarriers on tumor blood vessels have been reported in 2009 for mice models of metastatic colon cancer [49]. These studies have demonstrated reduction of the tumor microvascular index by 40%, extensive vascular occlusion and necrosis, and increased tumor permeability (however, complete tumor eradication could not be achieved in this particular case, because viable tumor cells remained around unaffected microvascular network). It should be mentioned that since 1910s researchers have noted that some colloids could accumulate in solid tumors soon after intravenous administration thus causing extended vascular damage and tumor tissue necrosis [50,51], although at that time the reasons for these observations were unknown and the proposed theories were quite speculative and largely debated. From the present viewpoint these early observations can be somehow explained with the EPR effect and the specific physicochemical characteristics of the intratumoral microenvironment, however the intimate mechanisms of how various colloids, including the PSMA-based ones, can cause damage to tumor vasculature still remains unclear. One possible (but requiring confirmation) suggestion is that in addition to the EPR effect, some colloids might become unstable in the tumor microenvironment and form precipitates thus causing vascular thrombosis, followed by local tissue necrosis. 4.3. Efficacy ofPSMA-based formulations in cancer chemotherapy

The PSMA-conjugated neocarzinostatin (SMANCS) has been the first macromolecular drug based on PSMA to be approved for use in clinical settings in 1993 [18,31]. When hepatoma patients received intraarterial (i.a.) SMANCS in Lipiodol (i.e., in a combination with a lipid contrast agent to make it visible with computed tomography) researchers observed that the tumor/blood ratio of drug distribution increased more than 2000 times, and retention of Lipiodol could last for more than 2-3 months, which was associated with markedly regressed tumor and prolonged survival of patients [31,52].





Antitumor effects of PSMA-based formulation of zinc protoporphyrin IX (ZnPP) have also been reported [35,36]. These studies have achieved a remarkable cytotoxicity of ZnPP formulation against various tumor cells; average IC 50 has been found to be approximate 5fold higher for various normal cells in comparison with the tumor cells. However, one must keep in mind that side effects of cytostatic therapies are usually related with cytotoxicity against rapidly dividing normal cells, such as bone marrow stem cells, progenitor cells in rapidly renewing epithelia, etc. (because metabolisms of cancer cells and rapidly dividing normal cells share many common features). Unfortunately, most studies of potential anticancer drugs compare the cytotoxicity to cancer cells with that to normal fibroblasts, normal hepatocytes or normal epithelia, but rarely (if at all) to normal stem cells.

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Doxorubicin formulated as PSMA micelles has been found to be less cytotoxic against cancer cells in vitro than the free drug [32]. The association with PSMA also decreased dramatically the drug toxicity in vivo ; however the micelles could accumulate in the tumor tissue via the EPR effect. The same report has demonstrated quite high antitumor efficacy of the doxorubicin-PSMA formulation against tumor formed by subcutaneous injection ofS-180 sarcoma cells. Pharmacokinetic analysis have revealed that the PSMA-formulated doxorubicin possessed increased half-life in plasma with 25-fold increase in the area under the curve (AUC) and concentration in tumor was 13-fold higher than free drug in mice 24 h after i.v. administration [32]. Similar studies with PSMAformulated pirarubicin have indicated about 5-fold higher in vivo antitumor activity than the respective doxorubicin formulation [33]. The authors have declared a remarkable effect of complete tumor eradication in 100% of tested animals bearing S-180 subcutaneous tumors. Further studies have confirmed significant antitumor efficacy in vivo in liver metastatic models in mice [49,37]. It has been found that in these cases pirarubicin equivalent of 100 mg/kg given as PSMA formulation could reduce tumor volume by 80% and achieve a survival rate of 93% at 40 days after tumor inoculation [37]. These authors have revealed that after treatment with the pirarubicin-PSMA formulation tumors underwent heterogeneous necrosis, however necrosis was incomplete in some tumors with residual cells persisting at the tumor periphery thus compromising complete cure [49,37]. Metastatic cancer models thus appeared to be more difficult to eradicate in comparison with the subcutaneous implanted tumors. However, metastatic models are somehow more realistic and closer to real malignant neoplasia. In another study, tanespimycin (an anticancer agent with dose limiting hepatic and gastrointestinal toxicity) has been formulated as PSMA-based micelles in order to improve its therapeutic efficacy [34]. The cytotoxicity of PSMA-tanespimycin micelles has been found to be diminished in vitro as compared to the free drug. Lower cytotoxicity of colloidal formulations can potentially be advantageous in vivo by minimizing systemic toxicity, while allowing time for accumulation in tumor to occur via the EPR effect [34]. These authors have found that the PSMA-tanespimycin micelles possessed significantly higher anticancer efficacy in vivo as measured by tumor regression when compared to free tanespimycin against subcutaneously implanted human prostate cancer in mice.

5. Interaction with the immune system

The interaction of nanocarriers with the immune system is highly important for their efficacy in cancer treatment. Artificial drug nanocarriers injected into the blood circulation are actually foreign structures for the organism and are usually non-specifically recognized as such by the innate immune system. Nanocarrier systems usually become opsonized by plasma components (mostly proteins, like immunoglobulins, apolipoproteins, complement components, etc.) after entry into the blood and soon after become engulfed by phagocytic cells (granulocytes, tissue macrophages) [48]. However, the opsonization of nanocarriers is usually due to nonspecific adsorption of opsonins in comparison to the relatively specific recognition of microbial invaders. Many tissue macrophages are situated in the liver sinusoids (Kupffer cells) and spleen and the uptake of nanocarriers by these phagocytes is considered as the main reason for accumulation of nanocarriers in these organs after intravenous administration [48]. However, when accumulation ofnanocarriers in the liver and spleen is considered, one should keep in mind that the sinusoids in these organs are fenestrated (fenestrations in liver sinusoids are ~ 100 nm and much larger in the spleen) and nanocarriers could pass through these fenestrations thus additionally favouring the accumulation in these organs. Of course, nanocarriers can be engulfed also by the phagocytic cells in blood (neutrophils, monocytes). In order to achieve effective intratumoral accumulation via the EPR effect nanocarriers must be


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able to circulate for longer time in the bloodstream, which means that they must not be easily recognized by phagocytes. This can be achieved by the use of poly(ethylene glycol)-coated nanocarriers, which are known to adsorb proteins (incl. opsonins) to a lower degree [48]. Albumin, the most abounded blood plasma protein, is usually considered as anti-opsonin [14], therefore its adsorption on the nanocarrier surface may be considered beneficial for achieving longer circulation times of nanocarriers in the bloodstream. Indeed, it has been previously demonstrated that SMANCS [31,52] and PSMA-based micelles [32,33] interacted and formed stable complexes with albumin. In our experiments with pure and epirubicin-loaded PSMA nanoparticles, we observed a significant change in the zeta potential of particles from -30 mV to -15 mV after addition of human serum albumin, indicating a possible adsorption of this protein on the nanoparticle surface, thus causing changes in their electrokinetic behaviour [41]. Previous pharmacokinetic studies of PSMA-based micellar formulations have revealed increased accumulation of carriers in the liver and spleen [32,36]. This observation is typical for nanocarrier systems and is usually related with their uptake by macrophages in these organs [48]. The generally observed liver accumulation of nanocarrier systems has incited many researchers to suppose a potential application ofthese carriers for the treatment of liver cancers. For example, the observed liver localization of ZnPP-PSMA micelles has been supposed as a possibility to target liver cancer [36]. Similar was the situation with cytostatics-loaded PBCA nanocarriers [53], which motivated clinical trials with hepatocellular carcinoma [54]. However, polymeric nanocarriers have been found to be located within the liver phagocytic cells (Kupffer cells) but not in liver parenchyma, which must be taken into account when planning liver cancer treatment with polymer nanocarrier systems [55]. The interactions between nanocarrier systems and macrophages are not related only to the biodistribution profile, and thus to the pharmacokinetics of the drug carriers, but also to activation or blockade of the phagocyte function. It has been previously shown, that PSMA nanocarriers could confer immuno-potentiating activity in treated animals, which is in clear contrast to the immuno-suppression caused by conventional smallmolecular-size anticancer drugs [56-58]. One should keep in mind that activation of macrophages may be a general reaction of the innate immune system after entering of foreign colloidal substances in the bloodstream, although it remains to be evaluated for a

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number of materials. In some cases of polymer nanoparticles, such as PBCA, activation of macrophages also has been reported, although higher doses of nanoparticles could cause saturation and physical blockade of macrophages [59,60]. Also, accumulation of nanocarriers in macrophages may be toxic to these cells and eventually to the organs where these cells are situated, because of overloading with foreign and indigestible material (see section 6). Both physical blockade and/or toxicity against phagocytes are rather possible to cause significant decline in cell immunity (thus making the organism prone to infections), if nanocarriers are overdosed, and this should be tested in future preclinical trials for a variety of nanocarrier systems in order to optimize the dose regimes. 6. Toxicity

Almost all reports considering in vitro and in vivo tests of PSMA-based formulations have declared lack of toxicity within the tested therapeutic ranges [31-36]. In most of these studies authors have reported even decreased drug toxicity ( in vitro and in vivo ), which effect has been explained by slow intracellular uptake of the polymeric drug by endocytosis, in addition to a strong association between the polymeric carrier and the drug thus resulting in slow release of free drug from the polymeric carrier [32,34]. This has allowed increasing the doses up to 10fold the maximal tolerable dose of the free drug [32,33]. However, pharmacokinetic studies of some PSMA-based formulations indicated increased accumulation in the liver, spleen and kidneys when compared with the free drug [32,36]. In other types of polymer nanoparticles (such as cyanoacrylates) it has been demonstrated that the alteration of the drug biodistribution by its association with nanocarriers could change the drug toxicological profile. For example, it has been shown that the incorporation of doxorubicin in cyanoacrylate nanoparticles could reduce its cardio-toxicity, but increased the bone marrow toxicity [61]. Possibilities for such changes in the toxicological profile ofa drug after its association with nanocarrier systems must always be considered during in vivo tests of new nanopharmaceutical formulations. Bone marrow and liver toxicity, as well as histological examinations of various organs, are usually always considered in more comprehensive toxicological studies of polymeric nanocarrier systems [62]. Changes in the function of bone marrow are usually followed by





only into the tumor. However, the mechanism of toxicity and the type of cell death, generally necrosis and/or apoptosis [37], which nanocarriers may induce in cancer cells at toxic concentrations, is another issue that awaits to be solved in more details. Unlike necrosis, apoptosis produces cell fragments that are engulfed by phagocytic cells before the contents ofthe cell can spill out and cause damage [64-66]. It is important that cytotoxic drugs and nanocarriers do not cause significant necrosis (and lysis) of tumor cells. Massive lysis of tumor cells may cause the so-called tumor lysis syndrome, which is considered as a medical emergency and may have devastating effects on the patient, such as hyperkalemia (rising of plasma potassium levels, causing muscle and cardiac abnormalities), hyperphosphatemia, renal failure, etc. [67]. Therefore, it is desirable that cytotoxic drugs and nanocarriers can induce predominantly apoptotic cell death in cancer cells.

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evaluation ofblood counts, while liver functional tests are used as indicators of liver toxicity during efficacy studies. Changes in animal weight are usually used as an indicator of general toxicity. Such toxicological studies for PSMAformulated tanespimycin have revealed that animals treated with the PSMA-based formulation showed no significant difference in normalized mean animal weight during the studies (usually longer than 25 days) [34]. Blood counts, cardiac and liver function have been measured in mice receiving free anthracyclines (doxorubicin [32] and pirarubicin [33]) and animals receiving even higher equivalent dose of the PSMAformulated drugs. These studies have indicated significantly decreased haemoglobin level, leucopoenia, thrombocytopenia, increased levels of creatine phosphokinase and liver enzymes in the mice treated with free anthracyclines in comparison with the control group, while the animals treated with the PSMA formulations have not shown any difference in these indicators in comparison with the untreated control group [32,33]. One important issue when considering the potential toxicity ofa biomaterial used as a drug carrier system is its biodegradability. Indeed, conjugates of PSMA with other substances may be degraded in vivo via hydrolysis of the amide bonds. Half-esters of PSMA may also be degraded by hydrolysis of the ester bonds. The polymer backbone of PSMA copolymers however consists of strong carboncarbon covalent bonds and is not expected to be biodegradable. The main problem to be considered in such biomedical applications of synthetic polymer-based formulations is the overloading of phagocytic cells with excess of polymer material (as well as drug), which may lead to toxicity and a potential danger for physical blockade of the phagocytes by systematic high-dose administration ofnanoparticles [59,60]. Unexpected side effects, such as fever, bone pain, allergic reactions or other immune responses must always be considered when performing initial tests with a new polymeric drug delivery system. For example, such effects have been reported in a phase I trial with doxorubicinloaded cyanoacrylate nanoparticles [63]. There is a general agreement that the drug nanocarrier systems must not be toxic. This requirement is easily understood, especially if the drugs to be carried are relatively non-toxic. However, taking into account that most of the current cytostatics are actually cytotoxic compounds, the toxicity of the nanocarrier material itself in cancer chemotherapy might be even beneficial, if of course, this nanocarrier can be selectively accumulated

7. Conclusions

We can conclude that the PSMA-based nanopharmaceutical formulations (drug conjugates, micelles and nanoparticles) hold a great promise for improvement of cancer chemotherapy. The remarkable therapeutic efficacy demonstrated by the SMANCS formulation in clinical trials and PSMA-formulated anthracyclines (doxorubicin and pirarubicin) in subcutaneous and metastatic preclinical murine models should be regarded. However, some problems remain to be solved on technological, biomedical and regulatory level. We demonstrated that one of the technological problems, the fine control of the nanoparticle size distribution, could be solved by utilization of the nanoprecipitation method for preparation of PSMA nanoparticles. One of the main biomedical problems, the optimization ofdose regimes for various formulations and types of cancer, seems to be the greatest one and still remains to be solved. On the other hand, regulatory problems regarding clinical trails of new anticancer formulations should be solved, for example by optimization of the amount of required experiments and avoidance of unreasonable requirements for filing for drug approval. Acknowledgements

This work was financially supported by the Bulgarian


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Science Fund (project DMU 03/111). GY is also thankful to CMST COST Action CM1101. SEM images and DLS data were obtained with the technical help of Mr. Nikola Dimitrov and Dr. Mariana Boneva (Faculty of Chemistry and Pharmacy, Sofia University), respectively.

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Bulg. J. Chem. 2 ( 2014) 33-43

Надежда Ангелова, Георги Йорданов*

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Нанофармацевтични формулировки на основата на поли(стирен- съ -малеинова киселина) 1 Факултет по химия и фармация, Софийски университет “Св. Кл. Охридски”, бул. Дж. Баучер № 1, 1164 София, България * Автор за кореспонденция. E-mail: [email protected]

Получена: 27 март 2014 Редактирана: 26 април 2014 Приета: 29 април 2014 Излязла online: 12 май 2014

Съполимерите на стирена и малеиновата киселина с относително ниска молекулна маса притежават голям потенциал за приложение като компоненти на системи за лекарствено доставяне, особено в химиотерапията на ракови заболявания. Пионерските разработки в областта на приложението на тези полимери датират от 80-те години на 20-ти век, когато са направени първите макромолекулни конюгати на тази основа за селективно доставяне на лекарства до тумори. Към настоящия момент съществуват някои обещаващи формулировки на противоракови агенти на основата на тези съполимери, които са показали забележителна антитуморна активност при преклинични и клинични изпитания. Целта на настоящия обзор е да обобщи основните методи за получаване на мицели и наночастици от поли(стирен- съ-малеинова киселина) (PSMA), основните странтегии за доставяне на такива системи до солидни тумори, тяхната токсичност, фармакокинетика и ефикасност в преклинични и клинични изпитания. Очертани са настоящото състояние и бъдещите перспективи при използването на тези системи за лекарствено доставяне. Описани са и някои оригинални приноси на авторите относно получаването на наночастици от PSMA чрез нанопреципитация. Ключови думи: лекарствено доставяне, наночастици, полиалкилцианоакрилат.



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