Molecular Dynamics Simulations of Polyglycerol Dendrimers As Carriers for Haloperidol

MOLECULAR DYNAMICS SIMULATIONS OF POLYGLYCEROL DENDRIMERS AS CARRIERS FOR HALOPERIDOL: THEORETICAL AND EXPERIMENTAL RESULTS

Silva, A. O.1, de Queiroz, A. A. A.2

1, 2 Center for Research and Innovation in Advanced Materials biofunctional – Federal University of Itajubá – Unifei, Itajubá (MG), Brazil

The aim of this study was to prepare polyglycerol (PGLD) dendrimers conjugated with haloperidol (Hal) to give PGLD-Hal conjugates, a novel controlled delivery system through the intravenous (i.v.) route to reduce the frequency of administration, dose and adverse effects during the short-term management of manifestation of psychotic disorders. Experimental analysis of the PGLD-Hal showed that, on average, six haloperidol molecules were covalently bound to PGLD. In order to better understand the surface loading and distribution of the haloperidol molecules, molecular reactivitywas determined by evaluation of electronic properties using frontier molecular orbital theory (FMOT) and molecular dynamics simulations. It was shown that the surface loading and distribution of the haloperidol molecules was determined by both electronic effects and by the different dynamic conformations adopted by the modified dendrimer during the incremental addition of haloperidol. The analysis of the dynamic behavior of the PGLD-Hal generation 1.0 showed that its flexibility and its polarity changed with the incremental addition of haloperidol.

Key words: Polyglycerol dendrimer, Haloperidol, antipsychotic therapy, schizophrenia, bipolar disorder.

1. INTRODUCTION

Dendrimers are a class of polymeric compounds that can be distinguished from conventional linear polymers by their highly branched and symmetrical architecture [[1], [2]]. Due to their structural characteristics (nanosize dimensions, controlled size and shape, multifunctionality, globular topology and presence of internal cavities) makes them interesting candidates for applications such as drug delivery, and controlled release [[3], [4]].

The broad spectrum of applications being developed for dendrimers in areas such as microencapsulation, drug delivery, light harvesting, molecular recognition, and biocatalysis is a result of these remarkable features [[5]].

Dendrimers is now frequently used as drug carriers in medicine to improvethe therapeutic value of various water soluble/insoluble medicinaldrugs and bioactive molecules by improving bioavailability, solubility and retention time reducing the patient expenses, and risks of toxicity [[6], [7], [8]].

Encapsulation of medicinal drugs in dendrimers(nanomedicine) increases drug efficacy,specificity, tolerability and therapeutic index ofcorresponding drugs improving the protection of premature degradation and interactionwith the biological environment, enhancement of absorptioninto a selected tissue, bioavailability, retention time and improvementof intracellular penetration [[9], [10]].

Haloperidol (Hal) is an extensively used, highly potent antipsychotic drug.Haloperidol is a first-generation antipsychotic drug used for the treatmentof schizophrenia and more acutely in the treatment of acutepsychotic states [[11]].Haloperidol possesses a strong activity against delusions andhallucinations, most likely due to an effective dopaminergic receptorblockage in the mesocortex and the limbic system of thebrain.

Since the uninterrupted supply of antipsychotic medication therapy [[12]] is vital for patient health long-term drug delivery system would be an ideal candidate to improve drug adherence and to ensure a continuous supply of optimum dosage levels of the drug. The aim of our research is to understand under the perspective of the quantum chemistry the various factors that affect the crucial performance metrics of haloperidol-loaded water soluble dendrimers including- size, drug incorporation, loading and release.

Structure-based modeling and quantum chemistry studies[[13]] can be used to accurately understand the interactions between functionalized dendrimers and molecules of pharmaceutical and medicine interest. In this work, frontier molecular orbital theory coupled with molecular dynamics simulations were used to understand the structural features of the experimentally determined loading and distribution of covalently bound haloperidol and its impact on the biological properties of the PGLD dendrimer (Figure 1).

(A) /
(B) / (C)

Figure 1 – Illustration of chemical structures of PGLD (A), Haloperidol(B) and the conjugate PGLD-Hal (C).

1. MATERIALS AND METHODS

2.1PGLD-Hal synthesis

PGLD with generation 0(G0), 2(G2), 3(G4) and 4(G4) were synthesized by a modified step-by step allylation and dihydroxylation divergent synthesis reactions [[14]]. The PGLD with a number average molecular weight of 1.7 kDa and an average of 26 hydroxyls per molecule were synthesized in a step-growth was used for the haloperidol immobilization. The successful synthesis of PGLD G-3 (88% yield) was confirmed by 1H-NMR, 300 MHz, D2O; shift (, ppm): 4.8 (OH), 4.04 (OCH2-CH-CH2), 3.95-3.40 (CH2-O-CH2); 13C-NMR (75 MHz, D2O); Shift (, ppm): 62.9 (CH2OH, terminal unity), 82.0-81.5, 80.6-79.8, 74.7-73.9, 73.8-72.2, 71.8-70.7, 64.7, 63.1 (CH2-O-CH2, polyether backbone).

A Bruker Biflex (Bremen, Ge) matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (Maldi-Tof-MS) was used for the determination of isomolecularity of PGLD molecules. The energy source was nitrogen laser operating at wavelength of 337 nm and pulse width of 3 ns. The matrix used was indoleacrylic acid, which was prepared by forming a saturated solution in acetone. Then, a target plate was spotted with 5 L of dilute sample. The sample was allowed to completely dry before use. Standards were used to calibrate the instrument. The real mass of the PGLD G-3 determined from Maldi mass spectra was found to be 1,721.8 (Na+) (Figure 2). This value was consistent with the theoretical molecular weight of PGLD G-3 (1,689) reported by the literature [14].

Figure 2–MALDI-TOF mass spectra of polyglycerol G3.0 dendrimer.

Following recrystallisation from ethanol, Hal was suspended in acetone (~20 mL) and then heated under reflux with the acylchloride derivative of PGLD for approximately 24 hours. Et3N was employed as a base to scavenge HCl that was liberated from the reaction. The reaction was monitored by TLC until completion. The solvent was removed under vacuum leaving crude PGLD-Hal prodrug. The product was then purified by column chromatography using a silica gel stationary phase. The mobile phase was diethyl ether: methanol (95:5).The eluent was subsequently removed under vacuum, followed by overnight drying of the product at 37°C and clear amber-coloured oil was obtained.

The percent conjugation of Halwas determined by the1H NMRspectrum on the basis of the peak intensity ratio of themethylene protons of PGLD (OCH2CH2,  = 3.5 ppm) andthe two protons on the aromatic ring closest to the fluorineatom (= 8.1 ppm). Using this ratio, on average 56.8 +8 % of the PGLD end groups were conjugated to haloperidol molecules

2.2 In vitro properties

2.2.1. Determination of entrapment efficiency and drug loading in the PGLD

The Hal loading percent was determined by measuring the concentrationof free drug in aqueous medium. The aqueous medium wasseparated through dialysis (Sigma 3K30, Germany). The amount of Hal in the aqueous phase was estimatedby HPLC. The Halloading percentage was calculatedfrom Equation (1):

(1)

2.2.2. In- vitro release of haloperidol from PGLD

To determine the release rate of Hal from PGLD, 4 mLof aqueous solution of PGLD-Hal was added to the dialysis bags with amolecular weight cutoff of 12,400 Da and the sealed bags were placed in theglass test-tube with 30 mL of the phosphate buffer solution (PBS) 0.1 M (pH7.4) at 37 ºC containing 3% polysorbate 80 to provide sinking conditions with agitationof 150 rpm. One-milliliter aliquots were taken out of the dissolutioncells at pre-determined time intervals, replaced by freshPBS buffer and analyzed for released haloperidol by HPLC. Thecumulative%release profiles were obtained by taking the ratioof the amount of haloperidol released to the total drug contentin the same volume of sample.

2.2.3. Hydrophobicity of PGLD

To describe the hydrophobicity of PGLD, the binding constantof Rose Bengal to PGLD of different generation was used. Adsorption isotherms were measured in 0.1 M PBS (pH 7.4). A mass of 250 g/mL of PGLD were incubated with concentrations of 1–45 g/mL of dye solution for 4 h at 375°C. After dialysis for 1 hour the supernatants were determined photometrically at 542.1 nm. Control samples were used ineach experiment to omit the effect of binding of Rose Bengal to the dialysis bag, tubes and pipette tips. The binding constant Kb of Rose Bengal was calculated using Scatchard transformation according to Equation 2, where r is the amountof adsorbed dye, a is the Rose Bengal equilibrium concentration or free dye,and N is the maximum amount of dye bound.

(2)

The surface hydrophobicity or binding constant of Rose Bengal (Kb)was obtained from the slopes of the straight lines from a plot of Rose Bengalratio of the amount adsorbed and the free amount (r/a) versus adsorbed RoseBengal concentration (r). Repeated analysis (n=3) produced consistent slopes.

2.2.4. Study of the red blood cells hemolysis

The human fresh blood containing anticoagulant was centrifuged at1000g for 5 min. After discarding the plasma, erythrocytes were washed 3times with PBS to remove the serum proteins. The stock of erythrocytedispersion was usable within 24 hours at 6ºC. 100 μL of the erythrocyte dispersions were added to 1000 μL of PGLD-Hal, shaken and incubated at 37°C for 1 hour. To remove intacterythrocytes from debris the mixture was centrifuged for 3 min at 750 g and100 μL of the supernatant were added to 2000 μL of a mixture of absoluteethanol and concentrated HCl (40/1 (v/v) and centrifuged again. Theabsorption of the supernatant was measured by UV/Vis spectrophotometer at 398 nm[22]. No lysis (0%) and 100% lysis were taken as the control samples in PBSand deionized and bidistilled water respectively [23].

2.3.In silico properties

2.3.1. Determination of electronic properties by semi-empirical methods

The electronic properties of the PGLD-Hal were evaluated utilizing a PM3 semi-empirical method, with the Polak-Ribiere algorithm (a technique of conjugated gradient which uses one-dimensional searches) being used for the optimizations using Hyperchem 8.0program package [[15]].

2.3.2. Molecular dynamics simulation of PGLD-Hal

The PGLD-Hal structures were generated with hyperchem and saved in pdb format by Open Babel. The pdb file was used to make the molecular dynamics simulation inGromacsprogram for thethree generations of PGLD and to determine the log P by Marvin program. VMD program [11, 14] were used to visualize the results of the performed using the molecular dynamics Gromacs.The dendrimer system was prepared for molecular dynamics simulations using Desmond with explicit solvent [[16]]. The PGLD-Hal system was built using the SPC solvation model and the size of the box was determined automatically by creating a 10 Å, buffer zone around the dendrimer. The molecular dynamics simulation [[17], [18]], structure, minimization and relaxation steps were performed for 4.8 ns at 300K and 1.03 bar. Snapshot structures were recorded every 4.8 ps.

1. RESULTS E DISCUSSION

In spite of the reports on the preparation of dendrimers as drug carriers there is no study about PGLD-Hal on their theoretical results and biological safety for use as drug delivery systems. Meanwhile none of the prepared PGLD-Hal can circulate for a long time in the blood. For this purpose in the present study the experimental and theoretical of PGLD-Hal were prepared and evaluated for their biological safety.

Frontier Molecular Orbital Theory (FMOT) postulates that the interaction between the HOMO and the LUMO of molecules undergoing a reaction can provide a good approximation of reactivity [[19]]. A reaction will occur with increasing and often maximal overlap of the HOMO and LUMO orbitals. These electronic properties were calculated using PM3 method implemented in Hyperchem. For this study, the LUMO of the Haloperidol molecule and the HOMO of the dendrimer was determined. The difference in the energy between the dendrimer HOMO and Haloperidol LUMO was then determined and analyzed.

The HOMO of the PGLD was located on one of the terminal hydroxyl groups. This would allow an interaction with the LUMO in the Hal, which was found to be located on the Fluor group (Figure 3).

Figure 3– The HOMO orbital from a PGLD dendrimer portion of a hydroxyl end group (right) and the LUMO orbital of a Haloperidol molecule (left) a)Electrostatic potential map,b) HOMO, c) LUMO.

Figure 4 - Top - Increasing branches of PGLD dendrimer modified with one haloperidol submitted to semi-empirical calculations with Hyperchem to determine the HOMO energy value and its location (arrow). Bottom – HOMO-LUMO gap as defined by the HOMO energy value determined for each dendrimer structure and the LUMO of a haloperidol molecule (-4.61 eV);a) Electrostatic potential map, b) HOMO, c) LUMO.

These electronic property calculations were applied to increasing sizes of PGLD generation with and without Hal (Figure 4). This approach enabled confirmation that the position of the HOMO was in the terminal hydroxyl groups where the interaction with the LUMO of Hal was expected to take place. An important new observation was that when the first Hal molecule was covalently attached to the PGLD, the HOMO was always located on the opposite branch to the one with the zero length ether bonds between the dendrimer and the haloperidol.

The gap between the HOMO of the PGLD and the LUMO of the haloperidol decreased as the size of the dendrimer molecule increased (Figure 4,Table 1). According to the FMOT, the smaller the gap between the HOMO and LUMO the more likely is the reaction to take place. By analogy, it was expected that this gap would be even smaller if the whole PGLD was considered.

Figure 5 - Dependence of the HOMO-LUMO of the PGLD generation number.PGLD G2 (left) and PGLD G3 (right);a) Electrostatic potential map, b) HOMO, c) LUMO.

A detailed analysis of the dendrimer segments showed that the energies of both the HOMO and LUMO, with three haloperidol molecules attached was almost double the LUMO energy value of the haloperidol molecule. This would make the addition of a fourth drug to the dendrimer section under study very unlikely. Therefore, the theoretical maximum loading of a generation 3.0 PGLD with Haloperidol was found to be 12. However, electronic effects on their own are insufficient to completely understand the loading and distribution of the haloperidol molecules on the surface of PGLD. Observation of the position of the HOMO, and defining both the location and availability of the terminal group, was necessary to better understand the loading and distribution of the haloperidol molecules.

Table 1–Average values of the HOMO energy determined for the PGLD studied, and differences between the HOMO of the dendrimer of generation 3.0 and the LUMO of the haloperidol (-9.67 eV).

Energia HOMO (eV) / Energia LUMO (eV) / HOMO-LUMO (GAP)(eV)
Poliglicerol G1 / -10,45124 / 2,163702 / -12,614942
Poliglicerol G2 / -10,4185 / 2,097654 / -12,516154
Poliglicerol G3 / -10,33021 / 2,054429 / -12,384639
Haloperidol / -9,199816 / -0,6687754 / -8,5310406
H1_PGLD G1 / -9,026786 / -0,4633388 / -8,5634472

The addition of the first haloperidol is favorable because the HOMOs of the hydroxyl groups are available. After one haloperidol has covalently bound to one of the end groups, the HOMO is again located on a peripheral hydroxyl group making the addition of a second haloperidol favorable. When two drug molecules are bound to the dendrimer, it becomes much less likely that the addition of third haloperidol will take place because there is only a 50:50 chance that the HOMO will be available for interaction with a hydroxyl end group.

The addition of a fourth haloperidol molecule is very unlikely to occur within the same PGLD. Thus, the conjugation of additional haloperidol molecules requires overcoming increasingly higher energy barriers with available positions for conjugation being available for only half of the time. Additional conjugation reactions of haloperidol on the whole dendrimer are very unlikely because the energy gap and the overlap between the LUMO of glucosamine and the HOMO of the newly formed conjugate are large, and the carboxylic acid end group is not available. These molecular modeling based observations are consistent with the experimental data that averages of 6 haloperidol molecules were conjugated to the surface of a generation 3.0 PGLD dendrimer.

3.1 Molecular dynamics simulation analysis of PGLD dendrimers

To better understand the dynamic behavior of these dendrimers, molecular dynamics simulations were carried out for 4.8 ns. Molecular properties including the gyration radius and RMSD were estimated and plotted as a function of time using the VegaZZ trajectory analysis tool (Fig. 6). The molecular properties determined for the generations of dendrimers PGLDs G1, G2 and G3, a trajectory of 4.8 nm were determined in water at temperatures of 298, 310 and 315 K.

Figure 6 – Molecular properties determined for a generation PGLD G1, G2 and G3 dendrimer along atrajectory of a 4.8 ns. Root mean square (RMSD) in Angstroms.

The RMSD analysis, as a measure of flexibility and dendrimer folding [[20]-22], was conducted for all of the PGLD structures. A plateau was seen after the first 200 ps (Fig. 6). This means that the molecule explored the conformational space with similar differences when compared to the initial structure set as reference for the RMSD calculations. However, further changes in the explored conformational space were observed as indicated by the increase of the RMSD values towards the end of the simulation. Although the RMSD reached a plateau after 200 ps, RMSD values of approximately 13 Å indicated a flexible structure. This plateau was consistent with the profile that was obtained for the gyration radius where, after only at about 900 ps, limited fluctuations occurred. These fluctuations indicated that the overall shape of the structure was not undergoing major changes during the simulation period.

Figure 7shows the haloperidol release profiles fromPGLD dendrimershaving a drugcontent of 2%. The drug release profile from PGLD dendrimerscan bedivided into four zones: (i) initial burst period, during whichthe surface drug is dumped into the release medium; (ii) induction period, during which the drug isreleased at a gradually decreasing fast rate and (iii) slow releaseperiod, during which the drug is released at a steady slow rate;(iv).

The increase in dendrimergeneration influences theabsolute release profiles such that both, the cumulative amount ofdrug released at any time (including initial burst) and the inductionperiod increases. The increase in generation increasestheamount of drug close to the surface as well as the drug in the coreof dendrimer. The former is responsible for an increased initialburst while the latter causes an increase during the inductionperiod.

For the cumulative haloperidol release profiles, the increasein the drug released is offset by the increase in the total amountof drug contained in the particles. The final effect on releaseprofile is determined by the larger of the above-mentioned ratios.The drug released during initial burst is predominantly the druglocated close to the surface. For our system, the slight decrease ininitial burst on increasing the drug content probably happens dueto uneven drug distribution inside the particles.