- Open Access
Cationic nanoparticles for delivery of amphotericin B: preparation, characterization and activity in vitro
© Vieira and Carmona-Ribeiro; licensee BioMed Central Ltd. 2008
Received: 31 January 2008
Accepted: 07 May 2008
Published: 07 May 2008
Particulate systems are well known to be able to deliver drugs with high efficiency and fewer adverse side effects, possibly by endocytosis of the drug carriers. On the other hand, cationic compounds and assemblies exhibit a general antimicrobial action. In this work, cationic nanoparticles built from drug, cationic lipid and polyelectrolytes are shown to be excellent and active carriers of amphotericin B against C. albicans.
Assemblies of amphotericin B and cationic lipid at extreme drug to lipid molar ratios were wrapped by polyelectrolytes forming cationic nanoparticles of high colloid stability and fungicidal activity against Candida albicans. Experimental strategy involved dynamic light scattering for particle sizing, zeta-potential analysis, colloid stability, determination of AmB aggregation state by optical spectra and determination of activity against Candida albicans in vitro from cfu countings.
Novel and effective cationic particles delivered amphotericin B to C. albicans in vitro with optimal efficiency seldom achieved from drug, cationic lipid or cationic polyelectrolyte in separate. The multiple assembly of antibiotic, cationic lipid and cationic polyelctrolyte, consecutively nanostructured in each particle produced a strategical and effective attack against the fungus cells.
In the recent years, much work has been devoted to characterize nanoparticles and their biological effects and applications. These include bottom-up and molecular self-assembly, biological effects of naked nanoparticles and nano-safety, drug encapsulation and nanotherapeutics, and novel nanoparticles for use in microscopy, imaging and diagnostics . Particulate drug delivery systems such as polymeric microspheres , nanoparticles [3, 4], liposomes [5, 6], and solid lipid nanoparticles (SLNs)  offer great promise to achieve the goal of improving drug accumulation inside cancer cells without causing side effects. Particulate systems are well known to be able to deliver drugs with higher efficiency with fewer adverse side effects [6, 8]. A possible mechanism is increase of cellular drug uptake by endocytosis of the drug carriers [9–11]. The emergence of the newer forms of SLN such as polymer-lipid hybrid nanoparticles, nanostructured lipid carriers and long-circulating SLN may further expand the role of this versatile drug carrier aiming at chemotherapy with cancer drugs . Recently, new nanoparticulate delivery systems for amphotericin B (AmB) have been developed by means of the polyelectrolyte complexation technique [13, 14]. Two oppositelly charged polymers were used to form nanoparticles through electrostatic interaction as usual for the Layer-by-Layer approach (LbL). This approach creates homogeneous ultrathin films on solid supports based on the electrostatic attraction between opposite charges . Consecutively alternating adsorption of anionic and cationic polyelectrolytes or amphiphiles from their aqueous solution leads to the formation of multilayer assemblies .
On the other hand, some double-chained synthetic lipids such as dioctadecyldimethylammonium bromide (DODAB) or sodium dihexadecylphosphate (DHP) self-assemble in aqueous solution yielding closed bilayers (vesicles) or disrupted vesicles (bilayer fragments, BF, or disks) depending on the procedure used for dispersing the lipid . DODAB, in particular, bears a quaternary ammonium moiety as cationic polar head, which imparts to this cationic lipid outstanding anti-infective properties . Both amphotericin B and miconazole self-assemble and solubilize at hydrophobic sites of DODAB or DHP bilayer fragments in water solution exhibiting in vivo therapeutic activity [19–22]. Over the last decade, our group has been describing the anti-infective properties of cationic bilayers composed of the synthetic lipid dioctadecyldimethyl ammonium bromide (DODAB) [17, 18, 21–27]. Adsorption of DODAB cationic bilayers onto bacterial cells changes the sign of the cell surface potential from negative to positive with a clear relationship between positive charge on bacterial cells and cell death . Regarding the mechanism of DODAB action, neither bacterial cell lysis nor DODAB vesicle disruption takes place . Recently, it was shown that the critical phenomenon determining antifungal effect of cationic surfactants and lipids is not cell lysis but rather the reversal of cell surface charge from negative to positive . In this work, we combine the SLN and the LbL approaches to develop novel and effective cationic particles to deliver AmB to C. albicans. Cationic microbicides self-assemble in a single supramolecular structure. The first attack against the fungus comes from an outer cationic polyelectrolyte layer. Thereafter the inert carboxymethylcellulose (CMC) layer is unwrapped so that monomeric AmB solubilized at the edges of DODAB bilayer fragments (BF) and the BF themselves can contact the fungus cell. Maybe this design represents a very effective cocktail against multidrug resistance. Complete loss of fungus viability could not be achieved before at the same separate doses of each component.
Results and Discussion
Colloid stability and antifungal activity of cationic bilayer fragments/amphotericin B/carboxymethyl cellulose/poly(diallyldimethylammonium) chloride at low drug-to-lipid molar proportion
Sizing and zeta-potential of drug, cationic lipid and anionic polyelectrolyte in separate or as assemblies
D ± δ (nm)
ζ ± δ (mV)
AmB in water
433 ± 5
-26 ± 3
- - -
79 ± 2
41 ± 2
79 ± 1
42 ± 2
88 ± 1
40 ± 1
145 ± 1
32 ± 2
90 ± 2
-50 ± 2
AmB in water
360 ± 4
-26 ± 3
AmB in IGP
75 ± 2
-27 ± 1
DODAB BF in IGP
- - -
75 ± 1
40 ± 1
195 ± 3
9 ± 1
199 ± 1
16 ± 1
1280 ± 80
4 ± 1
230 ± 2
-34 ± 1
The existence of bilayer fragments from synthetic lipids such as sodium dihexadecylphosphate, or dioctadecyldimethylammonium bromide or chloride obtained by sonication with tip has been supported by the following evidences: (i) osmotic non-responsiveness of the dispersion indicative of absence of inner vesicle compartment ; (ii) TEM micrographs with electronic staining ; (iii) cryo-TEM micrographs ; (iv) fluid and solid state coexistence and complex formation with oppositely charged surfactant ; (v) solubilization of hydrophobic drugs at the borders of DODAB bilayer fragments, which does not occur for DODAB closed bilayer vesicles [19–21, 33, 34]. They differ from the closed vesicles by providing hydrophobic borders at their edges that are absent in closed bilayer systems such as vesicles or liposomes. Under conditions of low ionic strength, due to electrostatic repulsion, the charged bilayer fragments remain colloidally stable in aqueous dispersions [19–21, 33, 34].
The physical properties of different dispersions such as size and zeta-potential are given in Table 1 both at low and high P. The drug in water exhibits substancial aggregation (Dz = 360–433 nm), as expected from its hydrophobic character. The drug particle presents a negative zeta-potential of -26 mV explained by dissociation of its carboxylate moiety at the pH of water . Upon changing the medium to IGP buffer, as previously reported, a decrease in size for AmB aggregates was observed (Dz = 75 nm) (Table 1), due to the chaotropic (dispersing) effect of dihydrogenphosphate anion on AmB aggregates . Both types of AmB aggregates interacted with DODAB BF yielding either loaded BF fragments at low P or DODAB covered drug particles at high P. The characteristics of these cationic assemblies before and after their interaction with oppositely charged CMC over a range of concentrations (0.001–1.0 mg/mL) are in Table 1. At low P, charge reversal took place above 1 mg/mL CMC whereas at high P, it occurred above 0.1 mg/mL CMC (Table 1).
Sizing, zeta-potential and antifungal activity of drug, cationic lipid, and polyelectrolyte(s) assemblies
Cationic lipid, drug and polyelectrolyte assemblies
D ± δ (nm)
ζ ± δ (mV)
DODAB BF (0.6)/AmB (0.005)/CMC (1)/PDDA(1)
171 ± 1
24 ± 2
79 ± 5
DODAB BF (0.6)/AmB (0.005)/CMC (1)/PL5000–10000 (1)
92 ± 4
40 ± 1
71 ± 4
DODAB BF (0.6)/AmB (0.005)/CMC (1)/PL30000–70000 (1)
138 ± 5
50 ± 3
21 ± 9
DODAB BF (0.6)/AmB (0.005)/CMC (1)/PL70000–150000 (1)
148 ± 5
60 ± 3
13 ± 5
AmB (0.05)/DODAB BF (0.06)/CMC (0.1)/PDDA (1)
280 ± 2
35 ± 1
27 ± 2
AmB (0.05)/DODAB BF (0.06)/CMC (0.1)/PL5000–10000 (1)
238 ± 1
25 ± 7
37 ± 1
AmB (0.05)/DODAB BF (0.06)/CMC (0.1)/PL30000–70000 (1)
326 ± 5
36 ± 3
23 ± 6
AmB (0.05)/DODAB BF (0.06)/CMC (0.1)/PL70000–150000 (1)
417 ± 3
47 ± 5
11 ± 3
The importance of large positive zeta-potentials for high efficiency of drug assemblies with DODAB BF and polyelectrolytes can be clearly seen from Figure 5. Negatively charged assemblies like those in Figure 5A and 5B yielded 100% of cell viability. Positively charged assemblies obtained upon increasing [PDDA] reduced cell viability to 50% (CMC/PDDA) (Figure 5C) or to 0% (DODAB BF/AmB/CMC/PDDA above 5 mg/mL PDDA) (Figure 5D). The schematic drawing in Figure 5D illustrates the layered assembly of microbicides in a single supramolecular assembly. The first attack comes from the outer cationic polyelectrolyte layer. Upon unwrapping this first layer and the second inert CMC layer, monomeric AmB contacts the fungus cell followed by the also effective DODAB action. Maybe this design represents a very effective assembly against multidrug resistance. Complete loss of fungus viability can seldom be achieved at the same separate doses of each component .
Colloid stability and antifungal activity of AmB/DODAB BF/CMC/PDDA at high P
At high P, 0.1 mM DODAB BF is sufficient to cover all AmB particles present in dispersion at 0.05 mM AmB with a thin, possibly bilayered, 6–8 nm DODAB cationic shell as previously described . This cationic interface is expected to interact with the oppositely charged CMC polyelectrolyte. At 0.1 mg/mL CMC, AmB/DODAB BF/CMC anionic complexes present high colloid stability, 230 nm mean diameter and -34 mV of zeta-potential (Figure 6C and 6D). This condition was chosen for further coverage with cationic polylectrolytes.
Fungizon (AmB in deoxycholate) and DODAB BF/AmB (formulation at low P) were previously evaluated in mice with systemic candidiasis . Both formulations yielded equivalent therapeutic results. However, DODAB BF/AmB was better from the point of view of reduced nephrotoxicity . Furthermore, cationic surfactants and polymers have an effect on integrity of red blood cells . Therefore, similar studies should be performed for the formulations described in this paper.
Optimal colloid stability and maximal fungicidal activity of monomeric or aggregated AmB in cationic lipid was achieved for cationic formulations at low or high drug to lipid molar proportions. At 0.005 mM drug, 1 mM DODAB, 1 mg/mL CMC and ≥ 5 mg/mL PDDA, monomeric AmB was found in DODAB BF enveloped by the two oppositely charged polyelectrolytes yielding 0% C. albicans viability. At 0.05 mM drug, 0.1 mM DODAB, 0.1 mg/mL CMC and PDDA ≥ 2 mg/mL, AmB/DODAB BF/CMC/PDDA assembly contained AmB in the aggregated state forming drug particles sequentially covered by DODAB BF, CMC and PDDA yielding also 0% fungus viability. The less tightly packed assembly turned out to be the one at high P, and high drug concentration which easily delivered the drug to cells at the lower zeta-potentials. The more tightly packed assembly was the one at low P, delivering drug to cells at higher zeta-potentials and lower drug concentration. In vitro both types of AmB formulations yielded complete fungicidal effect against Candida albicans (1 × 106 cfu/mL) representing good candidates to further tests in vivo.
Drug, lipid, polyelectrolytes and microorganism
Dioctadecyldimethylammonium bromide (DODAB), 99.9% pure was obtained from Sigma Co. (St. Louis, MO, USA). Carboxymethyl cellulose sodium salt (CMC) with a nominal mean degree of substitution (DS) of 0.60–0.95, poly(diallyldimethylammonium chloride) (PDDA) with Mv 100,000–200,000 and polylysines (PL) were obtained from Sigma (Steinheim, Germany) and used without further purification. Amphotericin B (AmB, batch 008000336) was purchased from Bristol-Myers Squibb (Brazil) and was initially prepared as a 1 g/L stock solution in DMSO/methanol 1:1. Candida albicans ATCC 90028 was purchased from American Type Culture Collection (ATCC) and reactivated in Sabouraud liquid broth 4% before plating for incubation at 37°C/24 h. In order to prepare fungal cell suspension for antifungal activity assays, three to four colonies were picked from the plate and washed twice either in isotonic glucose phosphate buffer (IGP; 1 mM potassium phosphate buffer, pH 7.0, supplemented with 287 mM glucose as an osmoprotectant) [39, 40] or in Milli-Q water by centrifugation (3000 rpm/10 minutes), pelleting and resuspension. The final fungal cell suspension was prepared by adjusting the inoculum to 2 × 107 cfu/mL and then diluting by a factor of 1:10 either in IGP or in Milli-Q water yielding 2 × 106 cfu/mL.
Preparation of lipid dispersion and analytical determination of lipid concentration
DODAB was dispersed in water or IGP buffer, using a titanium macrotip probe . The macrotip probe was powered by ultrasound at a nominal output of 90 W (10 minutes, 70°C) to disperse 32 mg of DODAB powder in 25 mL water solution. The dispersion was centrifuged (60 minutes, 10000 g, 4°C) in order to eliminate residual titanium ejected from the macrotip. This procedure dispersed the amphiphile powder in aqueous solution using a high-energy input, which not only produced bilayer vesicles but also disrupted these vesicles, thereby generating open BF [29, 41]. Analytical concentration of DODAB was determined by halide microtitration  and adjusted to 2 mM.
Determination of zeta-average diameter and zeta-potential for dispersions
Stock solutions of AmB were prepared at 1 mg/mL in 1:1 DMSO/methanol. Stock solutions of PDDA, CMC and PL were prepared at 20 mg/mL and diluted in the final dispersion to yield the desired final concentration. The stock solution of AmB (1 mg/mL) was added to DODAB BF dispersions to yield low and high drug to lipid molar proportions (P). At low P, dispersions contained final concentrations of drug, DODAB, CMC and PDDA equal to 0.005 mM (5 micrograms/mL), 1 mM (631 micrograms/mL), 0.01–2.00 mg/mL and 0.01–10.00 mg/mL, respectively. Firstly, DODAB BF and drug were allowed to interact for 10 minutes. Thereafter, CMC was added and allowed to interact for 20 minutes before adding PDDA, which was also allowed to interact for 20 minutes, before determining zeta-average diameter and zeta-potentials. At high P, a similar procedure was done this time at final concentrations of drug, DODAB, CMC and PDDA equal to 0.050 mM (50 micrograms/mL), 0.1 mM (63.1 micrograms/mL), 0.01–2.00 mg/mL and 0.01–10.00 mg/mL, respectively. At high P, drug particles were obtained at 0.050 mM AmB in IGP buffer yielding particles with 75 nm zeta-average diameter and -27 mV zeta-potential . These drug particles were firstly covered by DODAB BF and then wrapped by the polyelectrolytes over the quoted range of concentrations. Sizes and zeta-potentials were determined by means of a ZetaPlus Zeta-Potential Analyser (Brookhaven Instruments Corporation, Holtsville, NY, USA) equipped with a 570 nm laser and dynamic light scattering at 90° for particle sizing . The zeta-average diameters referred to in this work from now on should be understood as the mean hydrodynamic diameters Dz. Zeta-potentials (ζ) were determined from the electrophoretic mobility μ and Smoluchowski's equation, ζ = μη/ε, where η and ε are medium viscosity and dielectric constant, respectively. All Dz and ζ were obtained at 25°C, 1 h after mixing.
Optical spectra and aggregation state of AmB in the formulations
UV-visible optical spectra (280–450 nm) for characterization of AmB aggregation state were obtained in the double-beam mode by means of a Hitachi U-2000 Spectrophotometer against a blank of DODAB BF or DODAB BF/CMC (without drug), to separate light scattered by the dispersions from light absorption by the drug. All spectra were obtained at room temperature (25°C) at about 20 minutes after mixing DODAB BF and AmB at low or high drug to lipid P or after adding CMC to DODAB BF/drug assemblies.
Determination of cell viability for C. albicans ATCC 90028 as a function of polyelectrolytes concentration at low and high drug to lipid molar proportion (P)
At low or high P, DODAB/drug assemblies were wrapped by two layers of oppositely charged polyelectrolytes so that cfu were counted as a function of CMC and/or PDDA concentrations at 1 h of interaction time between C. albicans (1 × 106 cfu/mL) and formulations. Plating on agar plates for cfu counts was performed by taking 0.1 mL of a 1000-fold dilution in Milli-Q water of the mixtures. After spreading, plates were incubated for 2 days at 37°C. CFU counts were made using a colony counter. At low P, final DMSO/methanol concentration is 0.5% whereas at high P it is 5%. No effect of the solvent mixture at 0.5% on cells viability was previously detected . For further studies in vivo and at high P, it will be advisable to perform a dialysis step for the cationic nanoparticles aiming at complete elimination of the toxic solvent mixture.
This work was supported by the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and by the Fundação de Âmparo à Pesquisa do Estado de São Paulo.
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