Role of monensin PLGA polymer nanoparticles and liposomes as potentiator of ricin A immunotoxins in vitro

Journal of Controlled Release 50 (1998) 71–78

Role of monensin PLGA polymer nanoparticles and liposomes as potentiator of ricin A immunotoxins in vitro Abu J. Ferdous, Neena Y. Stembridge, Mandip Singh* College of Pharmacy and Pharmaceutical Sciences, Florida A& M University, Tallahassee, FL 32307 -3800, USA Received 24 January 1997; accepted 30 May 1997

Abstract Monensin is a carboxylic ionophore which can potentiate the activity of ricin based immunotoxins (IT) in vitro and in vivo against a variety of human tumours. Monensin was encapsulated into nanoparticles (NP) by using biodegradable poly( DL-lactide-co-glycolide) (PLGA, 50:50). The NP were prepared by modified emulsification-solvent evaporation method. High shear homogenization followed by simultaneous stirring and bath sonication were used for preparing NP. The size of NP was determined by photon correlation spectroscopy using a BI 90 particle sizer (Brookhaven Instruments). The average size of NP could be decreased from 567 nm to 163 nm by increasing the concentration of polyvinyl alcohol from 10% to 100% of PLGA. The NP were spherical in shape as observed by Atomic Force Microscopy. The concentration of monensin in the NP was analyzed by HPLC and the entrapment efficiency was found to be more than 12%. The zeta potential of NP was 225.8 (61.3) mv, which did not change significantly after resuspension of the freeze dried sample. The NP were tested against HL-60 and HT-29 human tumour cell lines in vitro. Monensin NP potentiated the activity of IT by 40 to 50 times against these cell lines. There was however, no difference between the NP and liposomes for their potentiating affect of IT against the two tumour cell lines.  1998 Elsevier Science B.V. Keywords: Monensin; PLGA polymer; Nanoparticles; Liposomes, Ricin A; Immunotoxin; Potentiator; HL-60 cells; HT-29 cells

1. Introduction Currently, various drug delivery systems are being used in cancer treatment and cure. One of the modalities being currently used is a combination of high dose chemotherapy and autologous bone marrow transplantation for eradication of Hodgkins lymphoma and several type of leukaemias [1]. However, the number of long term remissions is low

*Corresponding author. Tel.: 11 904 5612790; Fax: 11 904 5993347; e-mail: [email protected]

and patients who are cured face the risk of secondary malignancies and infertility. Another approach being currently used is treatment with immunotoxins (IT). Several ricin based ITs are presently in clinical trials, but all the reports so far indicate that these ITs need other drugs to potentiate their activity [2]. A major factor limiting IT efficacy is the lack of ricin A chain potency in vivo. Some antibiotics like cyclosporine in combination with IT have been used on limited number of patients to potentiate the anticancer activity of IT [3]. The anticancer drugs being used for IT potentiation are non-specific for the tumour cells and also act by

0168-3659 / 98 / $19.00  1998 Elsevier Science B.V. All rights reserved. PII S0168-3659( 97 )00116-8

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a mechanism different from IT. Presently, there is a dire need of non-toxic potentiating agents which can increase the activity of IT without increasing it’s toxicity. Monensin, a carboxylic ionophore has been successfully used as a potentiator of IT in vitro. However, it’s use in vivo is limited due to it’s lipophillicity. Liposomes have been used as a delivery system for targeting monensin to tumour cells. In our laboratory, successful experiments have been conducted with conventional monensin liposomes as potentiators of ricin based IT. The amount of liposomal monensin required for potentiation of IT is extremely low, 10 29 M, and produces a dramatic 20–30 times potentiation of IT in vitro against LOVO, LS 174T, H-meso, Gliomas and other tumour cells [4,5]. In vivo studies with H-meso tumours showed 30% long term survivors of mice treated with the antitransferrin receptor targeted IT and liposomal monensin [4,5]. In another animal tumour model using Namalwa cells, conventional monensin liposomes could increase the tumour kill efficiency of an CD 33 antigen targeted blocked ricin conjugate by 10 times [6]. Monensin liposomes were further conjugated to monoclonal antibodies to enhance their targetability. In vitro studies showed up to 40 times potentiation of the antitransferrin receptor targeted IT activity against LS 174T and H-mesothelioma human tumours [7]. Despite tremendous potential, the monensin liposome formulations have various problems and limitations which include, low drug entrapment efficiency, leakage and oxidation on long term storage and hydrolysis degradation of phospholipids [7,8]. The problems associated with the liposomal formulations can be overcome by delivering the drug in biodegradable polymer nanoparticles for parenteral administration. Polylactic acid (PLA), polyglycolic acid (PGA) and copoly(lactic / glycolic acid) (PLGA) are widely used biodegradable polymers. Aqueous suspensions of polymeric nanoparticles have been used in various pharmaceutical applications like film coating [9,10], drug targeting [11] and sustained release [12]. The initial problem of the nanoparticles was their clearance by the mononuclear phagocyte system (MPS). The problem was overcome by using gangliosides, poloxamers and polyethylene glycol (PEG) [13–16].

2. Materials and methods

2.1. Materials Poly(DL-lactide-co-glycolide) (PLGA, 50 / 50, molecular weight 14 000, polydispersity index 1.41, inherent viscosity 0.26 dL g 21 ) was obtained from Birmingham Polymers, Inc. (Birmingham, AL). Monensin and polyvinyl alcohol (Av. Mol. Wt. 30 000–70 000; as determined by Low Angle Laser Light Scattering; 87% hydrolysed) were obtained from Sigma Chemical Co. (St. Louis, MO). Radioactive 3 H-monensin was a gift from Dupont NEN (North Billerica, MA). Methanol, acetonitrile, methylene chloride, water and acetic acid were HPLC grade (Fisher, GA). Vanillin and other chemicals were reagent grade. Deionized, distilled water was used throughout the study. All cancer cell lines were obtained from American Type Culture Collection (Rockville, MD). All tissue culture media were obtained from Sigma Chemical Co. (St. Louis, MO). The IT conjugate was a gift from Dr. Thomas Griffin, University of Massachusetts Medical Centre (UMS), Worcester, MA (presently in Hoffman La Roche). The ricin A chain of the IT was produced from recombinant E. coli, and has an amino acid sequence identical to the chain A of native ricin, but is not glycosylated. It was disulphide linked to a native ricin iminothiolan derivatized mouse monoclonal IgG1 antibody (454-A) directed against the human transferrin receptor [7]. This conjugate was originally produced by Cetus Corporation (Emeryville, CA).

2.2. Preparation of nanoparticles and liposomes The emulsification-solvent evaporation method [15,17], with some modifications, was used to prepare monensin nanoparticles using biodegradable PLGA polymer. Initially 200 mg of copolymer PLGA and 20 mg of monensin were dissolved in 25 ml acetone. Two hundred mg of polyvinyl alcohol was dissolved in 50 ml distilled water. The polymer solution containing monensin was added to the aqueous phase drop wise and the mixture was homogenized with a Virtishear homogenizer (Virtis Company, Inc., NY), at 20 000 rpm for 20 min at

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low temperature. The emulsion was then simultaneously stirred (at 500 rpm) and sonicated in a bath sonicator for 1 h. The organic solvent was evaporated by gentle stirring using a magnetic stirrer for 24 h. Finally, the nanoparticles were washed and concentrated by centrifugation using Centriprep (Amicon Inc., MA) at 30003g for 2 h. The process was repeated three to four times until there was no monensin in the washings. Small unilamellar vesicle (SUV) liposomes containing monensin were prepared with the lipid composition dipalmitoylphosphatidylcholine: cholesterol: stearylamine (DPPC: CHOL: SA, 5:3:1) with an already published method [7]. The liposomes contained radioactive 3 H-monensin which could be easily analyzed by using an LKB Wallac 1219 Rackbeta Liquid Scintillation Counter (Wallac Inc., MD) [7].

raphy (HPLC) method developed in our laboratory was used for the analysis of monensin in the nanoparticles [18]. The HPLC system (Beckman, System Gold) consisted of a double pump Model 125, a visible detector 166 and an autosampler 507e. The mobile phase was methanol: acetonitrile: methylene chloride: water: acetic acid (45:20:25:9.5:0.5). The reverse phase C18 column was used and the flow rate was 1 ml min 21 . Monensin was reacted with vanillin reagent in the Beckman post-column reactor. The vanillin reagent was prepared by dissolving 8 g of vanillin in 100 ml of cooled methanol and 4 ml concentrated sulphuric acid. The post column reaction was carried out at 708C, where the reagent reacted with monensin and formed a pink colour, which was detected at 520 nm.

2.3. Particle size analysis

The total amount of monensin entrapped in each nanoparticle preparation, both before and after final washings was determined by HPLC [18]. The encapsulation efficiency was calculated based on the percentage of monensin entrapped in each batch of nanoparticle formulation in comparison to the starting amount.

The mean diameter and the standard deviation of the nanoparticles and liposomes were determined by photon correlation spectroscopy with a BI 90 particle sizer (Brookhaven Instruments Corp., Holtsville, NY). The particle size was analyzed after final washing. Nanoparticles and liposomes with an average diameter of about 160 nm were tested for their efficacy in cytotoxic studies.

2.4. Zeta potential analysis Zeta potential of each nanoparticle formulation was determined by Zeta Plus, zeta potential analyzer (Brookhaven Instruments Corp., Holtsville, NY). Zeta potential was measured by diluting a sample of formulation 1:16, with distilled water. An average of five readings were recorded. Zeta potential of each sample was analyzed at different stages: (a) after final washing of nanoparticles, (b) after adding 2% mannitol and 1% sodium CMC as cryoprotectant, prior to freeze drying, and (c) finally, after reconstituting the freeze dried product.

2.5. Determination of monensin by HPLC A modified high performance liquid chromatog-

2.6. Encapsulation efficiency of nanoparticles

2.7. Freeze drying of nanoparticles Samples of NP were freeze dried by using a ATR FD 3.0 freeze dryer (Appropriate Technical Resources, Laurel, MD). The system comprised of a 12 port floor model which was connected to a secondary vacuum trap. A Fisher Maxima pump (Model D8C) was used for vacuum. Prefrozen samples were placed in the flasks and connected to the ports of the freeze dryer. The samples were freeze dried within a few hours.

2.8. Atomic force microscopy (AFM) The nanoparticle images were taken with an Atomic Force Microscope (AFM) using a Nanoscope III (Digital Instruments, Santa Barbara, CA). Initially a drop of diluted nanoparticle suspension was placed on a glass slide and the sample was dried in a vacuum desiccator overnight. The images were

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scanned by tapping mode using a cantilever which oscillated vertically with a piezoelectric driver at a frequency of 350 kHz. The NP sample was viewed at 908 angle, scan size was 1 mm, setpoint 1.941 V, and scan rate was 1.209 Hz.

2.9. In-vitro potentiation of IT against different cancer cell lines The dye uptake assay using crystal violet was utilized to determine the cytotoxicity of all the formulations against HT-29 tumour cell line. Briefly, 6000 cells (HT-29) per well (100ml) were plated in a 96 well plate and allowed to settle overnight as a monolayer. Various dilutions of the IT were added along with a constant (non-toxic) amount of monensin nanoparticles. The cells were allowed to incubate in an incubator for three days, after which they were fixed in glutaraldehyde (0.25%) and then washed in running water. The cells were then stained with crystal violet and the excessive stain removed by washing with water. The plates were read by using a 7620 Microplate Reader (Cambridge Technology, Inc., MA) at an OD of 540 nm. From the absorbances of the treated cells and control, the percentage of cells killed was calculated. The values thus obtained were plotted against the concentration of IT. The TD 50 value was determined from the graph, which was the concentration of IT at which 50% of tumour cells were killed. Similarly, the MTT assay was used for suspension type cells (HL-60). For that purpose the MTT assay kit from Promega (Madison, WI) was used. Cytotoxicity of IT, IT in combination with blank nanoparticles, blank nanoparticles and antitransferrin antibody were also determined against the two tumour cell lines.

3. Results and discussion

3.1. Effect of stirring, sonication and homogenization The manufacturing method had a significant effect on the ultimate nanoparticle size. The effect of stirring, sonication and homogenization, either alone or in combination, on the nanoparticle size is shown in Fig. 1. Stirring, sonication or homogenization

Fig. 1. The effect of different process variables on the average diameter of nanoparticles. The stirring process was carried out at 500 rpm. Sonication was done using a bath sonicator at low temperature. Homogenization was carried out at 20 000 rpm at low temperature.

alone for one h produced NP of average size 3650 (6354), 2565 (6241) and 1087 (693) nm respectively. Stirring combined with sonication for one h produced NP of .1400 nm. When the three methods were combined together, i.e. the sample was homogenized, followed by simultaneous stirring and sonication, NP of size less than 200 nm was obtained. Finally, all batches of NP were produced by combining the three methods. During preparation, the homogenization process caused rapid diffusion of acetone across the interface and produced fine emulsion. Polyvinyl alcohol acted as a weak emulsifier in the process of emulsification. During the process, monensin was entrapped within the PLGA polymer. Sonication of the sample caused further reduction of particle size of the nanoparticles. During sonication, stirring was also done simultaneously which caused the evaporation of acetone and hardening of the PLGA polymer on the surface of the nanoparticles. These findings are in agreement

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with the results of Scholes et al. [17], who found that homogenization followed by probe sonication produced NP below 200 nm size. They observed that sonication greater than 5 min caused a rise in temperature, rapid solvent evaporation and subsequent increase in NP size. To avoid those problems, we used bath sonication at low temperature for one h, which produced NP smaller than 200 nm. Both probe and bath sonication were used to prepare nanoparticles. Though probe sonication was more powerful than bath sonication, the latter method produced smaller size NP than the probe sonication. This was due to the fact that probe sonication could not be carried out concurrently with stirring. Keeping other factors the same, probe sonication produced NP greater than 220 nm; whereas, bath sonication produced NP less than 200 nm. The size of nanoparticles is an important factor, because the formulation is intended for parenteral administration. Preparations containing particles greater than 250 nm are not suitable for direct intravenous injection. In preparing the nanoparticles, homogenization was followed by stirring and sonication, all of which were carried out at low temperature. Finally, the evaporation of organic solvent was carried out at room temperature. The method was developed in such a way, so as to avoid any degradation of the drug or biodegradable polymer during the manufacturing process.

3.2. Effect of polyvinyl alcohol ( PVA) concentration The concentration of PVA had a significant effect on the formulation of monensin nanoparticles. Increasing the PVA concentration from 20 mg to 200 mg (10–100% of PLGA) caused a decrease in average nanoparticle size from 567 (621) nm to 163 (66) nm (Fig. 2). A significant decrease in NP size was observed when PVA was increased from 20 to 50mg. Above 50 mg of PVA, the decrease in NP size was very small. Similar results have been reported by Scholes et al. [17]. They found that when PVA concentration was up to 20 times of PLGA, the NP size decreased, but above that concentration the NP increased in size. The increased viscosity of the formulation at the higher concentration of PVA caused an increase in size of NP. In our experiments,

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Fig. 2. The effect of PVA concentration on the average diameter of nanoparticles. Each batch was prepared in triplicate, and duplicate samples were analyzed for each batch.

the highest concentration of PVA was equal to PLGA concentration; therefore we observed that NP size decreased with the increase of PVA concentration.

3.3. Encapsulation efficiency, size and zeta potential analysis The percentage of monensin entrapped in NP varied with the size and is shown in Fig. 3. With the increase in NP size, the percentage of drug entrapped increased significantly. Similar findings have been reported by other workers [19] who found that the loading level for a protein in PLGA polymer microparticles increased from 1.2% to 5.1% when the average size of microparticles increased from 4.2 mm to 5.2 mm. In nanoparticle formulation, it was possible to entrap at least 12% of monensin. The amount of monensin entrapped in liposome (SUV) formulation, was found to be about 2% [6]. Therefore, by using biodegradable PLGA polymer, it was possible to entrap significant amounts of monensin in the NP. As the entrapment efficiency of NP is six times higher than the liposomal formulation, a smaller dose of nanoparticle formulation can be used for delivering the same amount of monensin. The zeta potential of the nanoparticles as de-

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cant change in monensin concentration in NP after reconstitution of the freeze dried product. Monensin liposomes were also studied for their stability at 48C. The size of the liposomes did not change by more than 5% after 12 weeks of storage. The amount of monensin which leaked from liposomes on storage was 7% [7].

3.5. Microscopic characterization of nanoparticles

Fig. 3. The percentage of monensin entrapped as a function of the NP diameter. Each batch was prepared in triplicate and the average concentration of triplicate samples were recorded.

termined after final washings was 225.8 (61.3) mv. The zeta potential of NP did not change significantly after resuspending the freeze dried sample. Similar findings have been reported by Calvo et al. [20] for polyester nanocapsules of cyclosporin A.

The morphology of NP was observed by atomic force microscopy (AFM), Fig. 4. AFM photographs showed that NP were uniform in shape, fairly monodispersed and without any aggregation. The surface of the NP appeared rigid and no monensin crystals were observed. The use of AFM for imaging PLGA and PEG-PLGA nanospheres has been reported by Gref et al. [14]. Scholes et al. [17] and Niwa et al. [21] studied the surface properties of PLGA nanospheres by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). They found that TEM was better than the

3.4. Effect of storage and drying The NP suspension in distilled water was stored at 48C for two months. The particle size analysis performed after that time showed that the average diameter of nanoparticles decreased from 163 nm to 156 nm. During storage, the leakage of monensin was less than 10%. The decrease in size can be attributed mostly due to surface erosion of PLGA polymer and to lesser extent on the release of drug from NP. AFM of the aged samples (not shown) revealed that no aggregation or clumping of the NP occurred during that time period. Samples of NP were freeze dried and particle size were analyzed after resuspending in distilled water. During freeze drying, 2% mannitol and 1% Sodium CMC were used as cryoprotectants. The average diameter of the NP increased from 171 nm to 187 nm. The 9% increase in particle size may be attributed to the aggregation of some smaller size particles during freeze drying. There was no signifi-

Fig. 4. Atomic force microscope (AFM) photograph of a sample of nanoparticles. A drop of diluted NP suspension was dried on a glass slide and the image was scanned by tapping mode. The cantilever oscillated vertically with a piezoelectric driver at a frequency of 350 kHz. The scan was viewed at 908 and the scan rate was 1.209 Hz.

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SEM for studying the nanospheres. But, both SEM and TEM techniques requires lengthy sample preparation process. Compared to SEM and TEM, AFM is a better and simple method. It is non-destructive, does not require any coating or other sample preparation technique and the sample can be viewed at it’s original form.

3.6. In-vitro potentiation of immunotoxin ( IT) against different cancer cells The potentiation effects of monensin nanoparticles and liposomes against HL-60 and HT-29 cancer cell lines have been summarized in Table 1. The results clearly show that monensin NP could potentiate the activity of IT by 50 times for HL-60 cells and by at least 40 times for HT-29 cells. There was no difference in potentiation for either monensin nanoparticles or liposomes. Similar potentiation was observed with monensin liposomes with IT against LS 174T, H-meso and glioma cell lines [4,7]. When either blank nanoparticles or liposomes were combined with IT, no significant change in cytotoxic effect was observed. Blank nanoparticles (or liposomes) and antitransferrin antibody when separately incubated with HL-60 and HT-29 cells were found to have no cytotoxic effect on these tumour cells (Table 1). The mechanism of action of monensin liposomes or nanoparticles for IT potentiation is very poorly understood. Transmission electron microscopic (TEM) studies performed with the HL-60 cells (not Table 1 Cytoxicity assay of monensin nanoparticles and liposomes with immunotoxins (IT) Cell line

IT IT1monensin nanoparticles IT1monensin liposomes IT1monensin (10 26 M) IT1nanoparticles (without monensin) IT1liposomes (without monensin) Blank nanoparticle (without monensin) Blank liposomes (without monensin) Antitransferrin antibody (500 mg)

TD 50 values (mg / ml) HL-60

HT-29

0.05 0.001 0.001 0.004 0.05 0.05 n.e. n.e. n.e.

2.0 0.05 0.05 0.075 2.0 2.0 n.e. n.e. n.e.

The amount of monensin in nanoparticles or liposomes510 29 M. n.e.5No effect.

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shown) incubated with monensin liposomes or nanoparticles (with IT) showed that at 30 min post incubation, the golgi of these cells were highly dilated which may be allowing the transport of the toxin molecules to ribosomal RNA. This was earlier demonstrated in our studies with CEM leukaemia cells [4]. More ultrastructure studies are being carried out to understand the exact mechanism of action of monensin formulations. This is the first publication on the preparation of monensin nanoparticles. Our laboratory has already standardized the preparation of monensin liposomes and it’s immunoconjugate. There was no difference in potentiation of IT by either nanoparticles or monensin liposomes. It appears that despite having different entrapment efficiencies and preparation methods, the uptake pathway or mechanism of action in vitro is the same for both of these formulations. Currently, we are preparing nanoparticles by the salting out method which will lead to the formation of a porous matrix and hence a very high entrapment efficiency. It is expected that these formulations may have a different release kinetics as compared to liposomes or the nanoparticles made by the solvent evaporation method. However, at this stage, the nanoparticle preparation seems to be a more viable approach due to it’s high stability and less leakage as compared to monensin liposomes. The nanoparticles were stable on storage and did not leak more than 10% of the drug after two months. In our experiments both liposomes and nanoparticles were of the conventional type and hence had a very short halflife. The choice of nanoparticles over liposomes is mostly due to it’s high entrapment efficiency, ease of formulation, and stability. We are also involved in the preparation of stealth monensin nanoparticles which will have a longer half life, so that the potentiation ability would be further augmented. The results will be further compared to stealth monensin liposomes which have already been standardized in our laboratory.

Acknowledgements The research was supported by RCMI award

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G12RR03020-11 and MBRS award 5S06GM0811124, both from NIH.

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