Elsevier

Biomaterials

Volume 26, Issue 11, April 2005, Pages 1299-1306
Biomaterials

Development and characterization of biodegradable nanospheres as delivery systems of anti-ischemic adenosine derivatives

https://doi.org/10.1016/j.biomaterials.2004.04.033Get rights and content

Abstract

We report a preliminary study concerning the encapsulation modalities in nanoparticles of the anti-ischemic drug N6-cyclopentyladenosine (CPA) and its pro-drug 5′-octanoyl-CPA (Oct-CPA). The release of these compounds and the related pro-drug stability effects in human whole blood have been tested. Moreover, the influence of the delivery systems on CPA interaction toward human adenosine A1 receptor has been analysed. The nanospheres were prepared by nanoprecipitation or double emulsion solvent evaporation method using poly(lactic acid) and recovered by gel filtration or ultracentrifugation or dialysis. Free and encapsulated Oct-CPA was incubated in fresh blood and its stability was analysed with HPLC. Quite spherical nanoparticles with mean diameters ranging between 210±50 and 390±90 nm were obtained. No encapsulation occurred when CPA was used. Satisfactory results concerning drug content (0.1–1.1% w/w) and encapsulation efficiency (6–56%) were achieved when Oct-CPA was employed. The controlled release of the pro-drug was achieved, being released within a range of 1–4 h, or very slowly, depending on nanoparticle preparations. The hydrolysis rate of Oct-CPA in human whole blood appeared stabilized in human whole blood with modalities related to the release patterns. The presence of all nanoparticle preparations did not interfere with CPA interaction at its action site.

Introduction

Adenosine is an endogenous ligand produced and released upon metabolic stress conditions [1], [2]. In the central nervous system it is able to hyperpolarize the neuronal membrane and to decrease the release of excitatory amino acids, showing a consequent neuro-protective action [1]. These physiological effects are mediated by the adenosine A1 receptor subtype, belonging to the family of adenosine receptors which have been classified as A1, A2A, A2B and A3 [3]. The activation of A1 receptors in the brain appears, therefore, a promising strategy in the aim to reduce ischemia-related structural and functional damages [4]. According to this point of view, very recently it has been observed that mice lacking the adenosine A1 receptors show a reduced protection against the neuronal degenerative effects caused by hypoxia [5].

In order to selectively reproduce the neuro-protective effects of adenosine, several ligands have been synthesized. N6-substituted adenosine derivatives and, in particular, N6-cyclopentyladenosine (CPA, Fig. 1) are currently identified as potent and selective A1 agonists [6]. This class of compounds can reduce, similarly to adenosine, the neuronal loss induced on brain slices in the presence of a hypoglycaemic medium [7], [8]. Moreover, CPA has been described to inhibit the ischemia-evoked release of excitatory amino acids into rat cortical perfusates [9]. In vivo studies have confirmed these results: selective A1 agonists have been, in fact, reported to protect gerbils from damage to the hippocampus and to increase survival following ischemic injury [10], [11].

Despite these encouraging laboratory results on the central nervous system, A1 agonists have not entered in the clinical use because of important side effects at other organs [11], [12]. These phenomena are attributed to the ubiquitous distribution of the adenosine A1 receptor subtype in the body [13]. Moreover, A1 agonists do not appear to have been absorbed into the brain [14] and can be quickly degraded in blood [15], [16].

Pro-drugs, as 5′-esters of CPA, were proposed to increase stability and diffusion through lipid barriers [17], [18]. Their hydrolysis was observed in whole blood and the lipophilic pro-drug 5′-octanoyl-CPA (Oct-CPA, Fig. 1) was found to be converted into CPA only by plasma esterases, with a relatively high rate (t1/2 about 30 min) [18]. It has also been demonstrated by us that CPA embedded into poly(lactic acid) microspheres displays a poor degradation in human whole blood [19], [20].

In order to obtain sustained release systems for CPA which can be potentially injected in vivo, we report here a study evaluating the encapsulation modalities of CPA and its pro-drug Oct-CPA in nanospheres, obtained using poly(lactic acid) polymer, which has been chosen for its good biodegradability and biocompatibility [21]. Different methods have been adopted for the preparation and recovery of nanoparticles and the related patterns about the release of CPA and Oct-CPA have been analysed. Finally, the effects of the release systems have been evaluated on the stability in human whole blood of the pro-drug Oct-CPA and on the interaction between CPA and the human adenosine A1 receptors.

Section snippets

Materials

[3H]DPCPX ([3H]1,3-dipropyl-8-cyclopentylxanthine, 108 Ci/mmol) was obtained from NEN Research Products (Boston, MA, USA). CPA, CHA (N6-cyclohexyladenosine), Pluronic F-68 (polyethylene-polypropylene glycols) and sulphosalicilic acid were obtained from Sigma (St. Louis, MO, USA). The pro-drugs Oct-CPA and CH-CPA (5′-cyclohexanoyl-CPA) were synthesized as previously described [18]. CHO cells transfected with adenosine A1 human receptors were a kind gift of Prof. Peter R. Schofield (Garvan

Nanosphere characterization

The CPA loaded nanoparticles obtained from the nanoprecipitation technique were discrete with a fine spherical shape and with an average diameter of 210±50 nm, while those prepared by the double emulsion solvent evaporation method were larger and more heterogeneous (mean diameter was 310±95 nm). However, the CPA amount in the nanoparticles was low (0.01% with the nanoprecipitation technique and 0.44% with the double emulsion solvent evaporation method), although an acceptable encapsulation

Discussion

The nanoparticle approach appears a promising strategy in the attempt to enhance the therapeutic effects of drugs potentially active in the brain [30], [31]. The anti-ischemic CPA and its pro-drug Oct-CPA appear as two attractive substrates for encapsulation studies in nanoparticle devices which could be suitable in order to protect them against a fast degradation [15], [16], [18] and, potentially, allow their transport and delivery into the brain. In this paper, we report a preliminary study

Conclusions

The results indicate that CPA encapsulation in poly(lactic acid) nanoparticles cannot be achieved under our experimental conditions, whereas promising results have been obtained by using the pro-drug Oct-CPA. This strategy has, in fact, allowed us to obtain the pro-drug sustained release and the decrease of its hydrolysis rate in human whole blood. Moreover, the presence of nanospheres does not interfere with the CPA interaction to its action site, i.e. the adenosine A1 receptor. These

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