| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
1
* Department of Pharmaceutical Sciences, University of Nebraska Medical Center, Omaha, Nebraska 68198, USA, and
Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, Nebraska 68198, USA
1Correspondence: 986025 Nebraska Medical Center, Omaha, NE 68198-6025, USA. E-mail: vlabhase{at}unmc.edu
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
|---|
Key Words: intracellular delivery • gene therapy • antiproliferative effect • sustained release • restenosis
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
|---|
, 2)
. Thus, in recent years there has been significant interest in developing carriers for intracellular delivery that will enable a variety of macromolecules to escape the degradative endo-lysosomal compartment and result in their efficient intracellular delivery (3
, 4)
.
Endo-lysosomal escape has been reported for a number of vectors used in gene therapy including viruses, fusogen peptides, cationic lipids, and cationic polymers. Viruses and peptide toxins use a fusogen peptide to cross the endosomal membrane and reach the cytosol (5)
. Nonviral vectors such as cationic lipids and polycations protect the DNA by either retarding the transfer of DNA from endosomes to lysosomes or destabilizing the endo-lysosomal membranes (2)
. However, these carriers suffer from a number of limitations including immunogenicity, toxicity, instability in vivo, and the ability to deliver only DNA or oligonucleotides (6)
.
A number of small protein domains, termed protein transduction domains (PTDs), can cross biological membranes without the necessity of endocytosis and have been shown to be useful for carrying various peptides and proteins into cells (7)
. However, these PTD vectors have a certain number of limitations in that they all require cross-linking to the target peptide or protein (8)
. Also, some of these systems such as PTDs derived from HIV-1 TAT protein require denaturation of the protein before delivery to increase the accessibility of the TAT-PTD domain (9)
. More recently, a short amphipathic carrier, Pep-1, was used to deliver functionally active proteins and peptides intracellularly without the need for cross-linking or denaturation (8)
. However, most of these vectors are also constrained by their ability to carry only protein or peptide therapeutics. Thus, there is a need for a carrier that is nontoxic and biodegradable and that has the ability to deliver intracellularly multiple classes of therapeutic agents.
We report here one such system, polymeric nanoparticles (NPs) formulated from the biodegradable polymer poly(DL-lactide-co-glycolide) (PLGA), that is able to cross the endosomal barrier and deliver the encapsulated therapeutic agents into the cytoplasm. NPs are colloidal systems that typically range in diameter from 10 to 1000 nm, with the therapeutic agent either entrapped into or adsorbed or chemically coupled onto the polymer matrix (10)
. The PLGA NP formulation with a therapeutic agent entrapped into the polymer matrix provides sustained drug release. The degradation products of PLGA are lactic and glycolic acids that are formed at a very slow rate and are easily metabolized in the body via the Krebs cycle and are eliminated (11)
. PLGA has previously been used for drug, protein, and gene delivery applications because of its biocompatibility and sustained-release properties (12
13
14
15)
. Thus, PLGA NPs offer the advantages of safety, the ability to carry different classes of therapeutic agents, and the possibility of sustained intracytoplasmic delivery. In this report, we demonstrate the rapid endo-lysosomal escape of PLGA NPs. We further demonstrate a sustained therapeutic effect of an NP-encapsulated low molecular weight drug with cytoplasmic receptor (dexamethasone) and sustained gene expression with DNA-loaded NPs as an example of a macromolecular therapeutic agent.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
|---|
NP formulation
NPs containing a fluorescent dye, 6-coumarin (fluorescent NPs), were formulated by using a double emulsion-solvent evaporation technique as described previously (16)
. In a typical procedure, a solution of bovine serum albumin (BSA) (60 mg in 1 ml of water) was emulsified in polymer (PLGA 50:50, molecular weight 143,000) solution (180 mg in 6 ml of chloroform) containing 6-coumarin (100 µg) using a probe sonicator (55 W for 2 min) (Sonicator XL, Misonix, Farmingdale, NY). BSA was used as a model macromolecule in the formulation of fluorescent NPs. The water-in-oil emulsion thus formed was further emulsified into 50 ml of 2.5% w/v aqueous solution of polyvinyl alcohol (PVA) used as an emulsifier by using sonication as above for 5 min to form a multiple water-in-oil-in-water emulsion. NPs containing plasmid DNA (pGL3 containing the firefly luciferase cDNA downstream of the SV40 promoter) were prepared by an identical procedure except that the DNA solution in Tris-EDTA buffer (0.6 ml, 10 mg/ml) was used to form the primary emulsion. NPs containing osmium tetroxide, an electron-dense agent, were formulated similarly, except that 10 mg of osmium tetroxide, instead of 6-coumarin, was added to the polymer solution. Dexamethasone-loaded NPs were formulated by emulsifying the polymer solution containing dexamethasone (50 mg of PLGA and 8 mg of dexamethasone dissolved in a mixture of 2 ml of chloroform and 0.5 ml of acetone) into a PVA solution (10 ml of 2.5% w/v) by sonication for 10 min as above to form an oil-in-water emulsion. In general, in all the formulation procedures, the emulsion was stirred for about 18 h at room temperature followed by desiccation for 1 h in a desiccator under vacuum to evaporate chloroform. NPs thus formed were recovered by ultracentrifugation (25,000 rpm for 20 min at 4°C, Optima LE-80K, Beckman, Palo Alta, CA), washed two times to remove PVA and unentrapped agent, and then lyophilized (Sentry, Virtis, Gardiner, NY) for 48 h to obtain a dry powder.
NP characterization
NPs were evaluated for size by TEM and for surface charge (zeta potential) by using a zeta potential analyzer (ZetaPlus, Brookhaven Instruments, Holtsville, NY). For TEM, a sample of NPs was suspended in water (0.5 mg/ml) and the particles were visualized by using a Philips 201 (Philips/FEI, Briarcliff Manor, NY) transmission electron microscope after negative staining of NPs with 2% w/v uranyl acetate.
Cell culture
Human arterial smooth muscle cells (HASMCs; Cascade Biologics, Portland, OR) were used for studying the cellular uptake of NPs, although human aortic vascular smooth muscle cells (HA-VSMCs, American Type Culture Collection [ATCC], Manassas, VA) were used for antiproliferative studies. VSMCs were selected for these studies because they have been implicated in the development of restenosis and have been used as an in vitro model for studying the antiproliferative efficacy of different therapeutic agents (17)
. HASMCs were maintained on Medium 231 supplemented with smooth muscle growth supplement (Cascade Biologics). HA-VSMCs were maintained on Ham’s F12-K medium supplemented with 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 10 mM N-tris[hydroxymethyl]methyl-2-aminoethane sulfonic acid (TES), 50 µg/ml ascorbic acid, 10 µg/ml transferrin, 10 µg/ml insulin, 10 ng/ml sodium selenite, and 30 µg/ml endothelial growth supplement, 10% FBS, 100 µg/ml penicillin G, and 100 µg/ml streptomycin (GIBCO BRL, Grand Island, NY). PC3 (prostate cancer) cells (ATCC) were grown in RPMI 1640 supplemented with 10% FBS, 100 µg/ml penicillin G, and 100 µg/ml streptomycin. We selected these cell lines because of our interest in developing NP-based drug and gene therapies for cancer and restenosis.
Cellular NP uptake studies
HASMCs were seeded at 50,000 cells/ml per well in 24-well plates (Falcon, Becton Dickinson, Franklin Lakes, NJ) and were allowed to attach for 24 h. To determine the NP uptake, the cells were incubated with a suspension of NPs in growth medium for 1 h, washed three times with PBS (pH 7.4, 154 mM), and lysed by incubating cells with 0.1 ml of 1X cell culture lysis reagent (Promega, Madison, WI) for 30 min at 37°C. The cell lysates were processed to determine the NP levels as per our previously published method (16)
. The fluorescent dye (6-coumarin) incorporated in PLGA NPs (
0.05% loading) is lipophilic and therefore remains associated with the polymer matrix of NPs. The dye does not leach from the NPs during the experimental period and therefore the fluorescence seen in the cells is due to NPs. Thus, the dye in NPs serves as a sensitive marker to quantitatively determine their cellular uptake and also to study their intracellular and tissue distribution by confocal or fluorescence microscopy (16
, 18)
.
Initially, the dose- and time-dependent cellular uptake of NPs was determined. To study the effect of various inhibitors on NP uptake, cells were preincubated first with inhibitors and then with a suspension of NPs (100 µg/ml), which also contained the respective inhibitor at the same concentration as that used for preincubation: 1) 0.1% w/v sodium azide and 50 mM 6-deoxyglucose for 1 h, 2) 10 mM ammonium chloride for 1 h, 3) 450 mM sucrose for 1 h, 4) 5 µM brefeldin A for 5 min, 5) 30 µM cytochalasin D for 30 min, 6) 33 µM nocodazole for 30 min, 7) 100 nM bafilomycin A1 for 30 min, and 8) 1 µg/ml filipin for 30 min. To study the effect of temperature on NP uptake, cells were preincubated for 1 h at 4°C and then with an NP suspension for an additional 1 h at 4°C. Confocal microscopy and TEM were used to monitor the cellular uptake and intracellular trafficking of NPs.
Microscopic studies
For confocal microscopy, HASMCs were plated 24 h prior to the experiment in Bioptechs plates (Bioptechs, Butler, PA) at 50,000 cells/plate in 1 ml of growth medium. To study the effect of various inhibitors on the intracellular distribution of NPs, the cells were first pretreated with inhibitors and then were incubated with a suspension of fluorescent NPs (100 µg/ml) containing 50 nM LysoTracker Red and the respective inhibitor for 30 min. For the experiments involving Texas Red transferrin, cells were preincubated with serum-free growth medium for 30 min followed by treatment with a suspension of fluorescent NPs (100 µg/ml) prepared in serum-free growth medium containing Texas Red transferrin (100 µg/ml). After the cells were incubated with NPs for 30 min, they were washed twice with PBS and visualized with HEPES buffer (pH 8). The images captured by use of a 488-nm filter (fluorescein), 568-nm filter (rhodamine), and differential interference contrast using a Zeiss Confocal microscope LSM410 equipped with argon-krypton laser (Carl Zeiss Microimaging, Thornwood, NY) were overlaid to obtain images to determine localization of NPs in endo-lysosomal compartments (using LysoTracker Red as a marker), or in early and recycling endosomes (using Texas Red transferrin as a marker).
For TEM, HASMCs were plated 24 h prior to the experiment in 100-mm tissue culture dishes (Becton Dickinson) at 500,000 cells/dish in 10 ml of growth medium. To study the time-dependent cellular uptake and intracellular distribution of NPs, the cells were incubated with the osmium tetroxide-loaded NPs (100 µg/ml), were washed twice with PBS at different time points (10 min, 30 min, and 1 h postincubation) for the time-dependent uptake study, and then were harvested by trypsinization. The harvested cells were fixed in a 2.5% glutaraldehyde solution in PBS and then postfixed in 1% osmium tetroxide in PBS for 1 h. The cells were washed with PBS and dehydrated three times in a graded series of ethanol solutions (50%, 70%, 90%, 95%, 100%); they were then soaked overnight in a 1:1 ratio of 100% ethyl alcohol and Unicryl embedding resin (Ted Pella, Redding, CA), after which they were soaked in fresh Unicryl resin for 4–5 h. The cells in fresh Unicryl resin were then placed in BEEM capsules (Electron Microscopy Services), and the capsules were placed in a Pelco UV-2 Cryo Chamber (Ted Pella) at 4°C for 48 h for polymerization of the resin by UV radiation. The polymerized blocks were sectioned, and the sections (80–100 nm thick) were placed on Formvar-coated copper grids (150 mesh, Ted Pella), stained with an aqueous solution of 2% uranyl acetate for 15 min, washed briefly in water, stained with Reynolds lead citrate for 7 min, and then finally washed in water prior to visualization under a Philips 410LS microscope (Philips/FEI).
In vitro antiproliferative studies
HA-VSMCs were plated overnight in 96-well plates at 2500 cells/well and had their growth synchronized by serum depletion for 24 h. Cells were then restimulated with normal growth medium containing 10% FBS. The growth medium also contained 10 µM dexamethasone either in solution or encapsulated in NPs. Plain growth medium and control NPs (no drug) were used as respective controls. Cell proliferation was followed by a standard MTS assay (CellTiter 96 AQueous, Promega).
In vitro gene expression studies
PC-3 cells were seeded in 24-well plates 24 h prior to transfection. The transfection was carried out at 60–70% confluency. The growth medium in the wells was replaced with a suspension of the DNA-loaded NPs prepared in the growth medium containing serum. Luciferase activity was measured by using an assay kit from Promega, and the data were normalized to per milligram of cell protein. The transfection study could not be continued beyond 3 days because by then the cells had reached full confluency and started to detach.
Statistical analysis
The two-tailed unpaired Student’s t test was used to analyze the significance of differences in mean responses between the various treatment groups. Differences were accepted as significant at P values < 0.05.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
|---|
A), with an average zeta potential of -12.5 ± 0.4 mV (mean ± SE, n = 5) at pH 7 in 0.001 M HEPES buffer (Fig. 1B
). The zeta potential and the particle size of the different NP formulations are shown in Table 1
.
|
|
Mechanism of NP uptake
The NP uptake by HASMCs was linear at lower doses of NPs (10–100 µg), but the efficiency of uptake was reduced at higher doses. The NP uptake was relatively rapid during the first 2 h of incubation, before a saturation uptake was achieved in 4–6 h. NP uptake was energy dependent, as evidenced by the reduction in the uptake by 78% and 67% at a lower temperature of incubation (4°C) and after energy depletion by a mixture of sodium azide and 6-deoxyglucose, respectively (Fig. 2
A). Inhibition of clathrin-coated pit endocytosis by hypertonic growth medium decreased the intracellular uptake of NPs by 40%, whereas inhibition of caveola-coated pit endocytosis by filipin did not affect NP uptake, indicating that caveolae were not involved in NP uptake (Fig. 2B
). NP uptake was not affected by cytochalasin D, a potent inhibitor of actin polymerization, which suggests that microfilaments did not play an important role in the uptake of NPs, at least in this cell line (Fig. 2B
). However, inhibition of microtubules by nocodazole resulted in a 66% increase in NP uptake, indicating that microtubules may be involved in controlling intracellular uptake of NPs (Fig. 2B
).
|
Intracellular distribution and endo-lysosomal escape of NPs
Because transferrin is a marker of early and recycling endosomes (19)
, we studied the colocalization of Texas Red-conjugated transferrin (red fluorescence) with fluorescent PLGA NPs (green fluorescence). This resulted in a partial colocalization (yellow fluorescence) in the peripheral cytoplasmic compartment (Fig. 3
A). However, NPs appeared to accumulate in compartments separate from the transferrin-labeled compartments, which suggests that a majority of NPs may be present either in late endosomes/lysosomes or in the cytoplasm. To determine whether the NPs are localized in the secondary endosomal and lysosomal compartments, cells were incubated with NPs in the presence of LysoTracker Red, a marker for secondary endosomes and lysosomes. The LysoTracker Red, which is colorless at physiological pH, has red fluorescence at the acidic pH present in these compartments. As shown in Fig. 3B
, NPs were colocalized with LysoTracker Red in the endo-lysosomal compartment within 2 min of incubation, as evident from the appearance of orange to yellow fluorescence. At 10 min postincubation, NPs were localized in the cytoplasmic compartment, as seen from the appearance of green fluorescence of NPs (Fig. 3C
). The intensity of green fluorescence in cytoplasm increased with incubation time, which suggests the localization of more NPs in the cytoplasmic compartment with time (Fig. 3D, E
). The above results thus indicate that NPs were probably escaping rapidly from the endo-lysosomal compartments into the cytoplasmic compartment following their uptake. This argument is supported by the fact that the brefeldin A-induced tubulation of secondary endosomes or lysosomes (20)
resulted in the lack of NP-associated green fluorescence in the cytoplasmic compartment. This result may be due to the inhibition of escape of NPs from the endo-lysosomal compartment to the cytoplasmic compartment because of the tubulation of endo-lysosomal vesicles (Fig. 4
). The above-mentioned events related to NP uptake and their cellular trafficking were then followed by using TEM. The osmium tetroxide-loaded NPs in the cells were clearly distinguishable from the intracellular vesicles because of their electron-dense nature. The TEM of the cells exposed to NPs demonstrated localization of NPs in vesicles (Fig. 3F
) and multivesicular bodies at 10 min postincubation, with few NPs in the cytoplasm. NPs were seen adhering to the wall of endocytic vesicles, which suggests some interaction between NPs and the membrane of the endocytic vesicles prior to the escape of the NPs into the cytoplasm (Fig. 3G
). With a further increase in the incubation time, more NPs were seen in the cytoplasmic compartment and in lysosome-like structures (Fig. 3H
). Thus, the TEM data complement the confocal data on NP uptake and their rapid escape from the endo-lysosomal compartment to the cytoplasmic compartment.
|
|
To determine the mechanism of NP escape from the endo-lysosomal compartment, we thought of various possibilities including the changes in the surface characteristics of NPs after their cellular uptake. We found that PLGA NPs have a negative charge in physiological and alkaline pH but acquire a positive charge in the acidic pH that is present in the endo-lysosomal vesicles (pH 4) (Fig. 1B
). Therefore, we considered the possibility that NPs may escape the endosomes by a mechanism similar to that operating for cationic lipids. To test this hypothesis, we incubated NPs in the presence of ammonium chloride, a lysosomotropic agent that is known to raise the pH inside the endosomal vesicles. This resulted in the complete escape of the NPs from the endo-lysosomes (Fig. 4A
). Incubation of NPs in the presence of bafilomycin A1 also led to similar results (data not shown), probably also related to the lysosomotropic effect of bafilomycin A1 (21)
. Although these results show that increasing the pH of the endo-lysosomes led to an increase in the number of NPs escaping into the cytoplasm, they did not elucidate the mechanism of escape of NPs into the cytoplasmic compartment in the absence of a lysosomotropic agent. Hence, we studied the intracellular distribution of fluorescent polystyrene NPs of similar size as PLGA NPs and without any surface functionalization. Unlike the PLGA NPs, these NPs exhibited a negative zeta potential in all pH values (Fig. 1C
). Almost all of the polystyrene NPs were found colocalized in the endo-lysosomal compartment, with most of the cytoplasmic compartment free of the green fluorescence of NPs (Fig. 4C
). The insignificant green fluorescence seen outside of the endo-lysosomal compartment can be attributed to the NPs localized in the early and recycling endosomes. These data support our hypothesis that surface cationization of PLGA NPs in the endo-lysosomal compartment is responsible for their escape into cytoplasm. This result is further substantiated by the fact that in the TEM pictures of the cells, PLGA NPs were clearly seen adhering to the membrane of endocytic vesicles (Fig. 3G
) but not in the early endosomes (Fig. 3F
), where the pH was 7 and the particles were negatively charged.
Sustained antiproliferative effect of dexamethasone-loaded NPs
The percent increase in the cell population as compared with the day 1 cell population of HA-VSMCs is shown in Fig. 5
. Dexamethasone in solution showed inhibition of cell growth compared with plain growth medium for only up to 4 days (55 ± 2% for growth medium vs. 45 ± 4% for solution) (Fig. 5A
). The inhibition of cell growth with dexamethasone NPs was significantly greater and more sustained (for up to 2 wk) as compared with the same dose of control NPs (Fig. 5B
) or dexamethasone in solution (Fig. 5C
). The inhibitory effect was not due to NPs, because there was no difference between the growth curves of the cell treated with either growth medium (Fig. 5A
) or control NPs (Fig. 5B
).
|
Sustained gene expression in prostate cancer cells
The transfection levels were dose dependent and increased with the time of transfection, which suggested sustained gene transfection with NPs (Fig. 6
). Similar experiments with a commercially available transfecting agent, FuGene 6 (Roche Diagnostics, Indianapolis, IN), resulted in a peak expression level at 2 days, which then declined by about 70% in 3 days. The plasmid DNA in solution alone showed an insignificant level of transfection.
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
|---|
. After intracellular uptake, the contents of the endocytic vesicle are delivered to lysosomes for degradation unless there are special mechanisms for the contents to escape out of the endo-lysosomes. Thus, it becomes critical in macromolecular delivery that the carrier used for intracellular delivery should also successfully protect the macromolecule from degradation in the lysosomes and deliver it to the cytoplasmic compartment (3)
. Further, for drugs such as dexamethasone, receptors of which are cytoplasmic, it may be important to retain the drug in the cytoplasmic compartment to enhance its therapeutic efficacy (22)
.
The efficacy of NP-encapsulated therapeutic agents such as low molecular weight drugs and macromolecules has been known (10
, 13
, 23)
, but the mechanism of their enhanced therapeutic effect has not been investigated at a cellular level. Although a therapeutic agent encapsulated in NPs may be less susceptible to degradation in the endo-lysosomal compartment, the relatively faster degradation of PLGA NPs under acidic conditions in the endo-lysosomal compartment (11)
may result in the release of the therapeutic agent in the endo-lysosomes, which could then degrade quite rapidly. We therefore hypothesized that to function as an efficient drug or gene delivery vehicle, NPs must be efficiently internalized into the cells and then deliver their payload into the cytoplasmic compartment rather than be retained in the degradative environment of endo-lysosomal compartment.
In our studies, PLGA NPs were internalized by VSMCs in an energy-dependent manner, which suggests an endocytic process (24)
. This result was further confirmed by the fact that NPs, once internalized, were found in the endosomal and lysosomal compartments. A similar energy-dependent and saturable uptake was reported for poly(ethylene oxide)-PLGA nanospheres in VSMCs, and it was suggested that the uptake was through adsorptive pinocytosis (25)
. NP uptake was significantly reduced after inhibition of clathrin vesicles but not caveolae. Clathrin-mediated transport has been previously reported for active receptor-mediated endocytosis (26)
. However, our NPs did not have any specific ligands for receptor-mediated intracellular uptake. Therefore, it is possible that NPs were being nonspecifically transported through clathrin vesicles. The fact that NPs were generally found in the center of the newly formed endocytic vesicle (Fig. 3F
) instead of being attached to the membrane wall provides further evidence that NPs were being internalized by a nonspecific mechanism, possibly by fluid-phase pinocytosis. VSMCs are known to be phagocytic, involved in acting on dead and apoptotic cells in the vessel wall (27)
. However, no phagocytic cellular activity was detected during the uptake of NPs at an NP dose as high as 1000 µg (assay kit: Fc-OxyBurst, Molecular Probes). Absence of any effect on the NP uptake after the inhibition of microfilament polymerization further confirmed that the NP uptake process is not phagocytic (24)
.
After their uptake, NPs were localized in the early and recycling endosomes and also in the late endosomes and lysosomes. We hypothesize that from early endosomes, NPs either are recycled back to the surface or are transported to the secondary endosomes and lysosomes from which the NPs escape into the cytosol (Fig. 7
). Similar sorting pathways at the level of early endosomes have been described for cell surface receptors that are either recycled back to the surface or degraded in the lysosomes (28)
. Endosomal escape has been reported for viral and nonviral vectors used in gene therapy (2
, 29)
. Although viral vectors use a fusogen peptide to cross the endosomal membrane, it is generally believed that the DNA-cationic compound fuses with the organelle membrane, leading to the escape of DNA into the cytosol. Another possibility is that the cationic lipids and polymers cause the swelling and rupture of the lysosomes by sequestering protons and their counterions (the "proton sponge effect") and create an osmotic imbalance similar to that created by lysosomotropic compounds (30)
. This latter mechanism could be the source of toxicity observed with the cationic vectors.
|
Although cationization of poly(lactide)or PLGA microparticles with the change of pH was previously reported and was attributed to the transfer of excess protons from the bulk liquid to the NP surface or attributed to hydrogen bonding between carboxyl groups of poly(lactide) or PLGA and hydronium molecules in the acidic pH (31
, 32)
, its significance in the escape of NPs from the endo-lysosomal compartment was not elucidated. This surface cationization could explain the differences in NP behavior in the different endocytic vesicles. The early endocytic vesicles have a physiological pH (24)
and at this pH, NPs would have a net negative charge and hence would be repelled by the negatively charged endosomal membrane. The TEM of the cells exposed to NPs showed NPs in the center of the early endosomes (Fig. 3F
), supporting the above argument. However, the secondary endosomes and lysosomes are predominantly acidic, with pH values ranging from 4 to 5 (24)
. In this pH, NPs would have a net cationic potential and hence would interact with the negatively charged membrane (Fig. 3G
), leading to their escape into cytoplasmic compartment. The escape of NPs is not due to the opening of the endo-lysosomal vesicles because no differences in the distribution of LysoTracker dye was found in the cells that were incubated with NPs for 24 h (data not shown). Lysosomotropic agents are known to cause the destabilization of endo-lysosomes because of the change in the pH of the endo-lysosomal vesicles, leading to the escape of their contents (21)
. Because of the release of the endosomal contents, lysosomotropic agents are known to have cytotoxic effects. Because we did not observe cellular toxicity with PLGA NPs in cell culture in a 48-h mitogenic assay in our previous studies (16)
, it is clear that NPs do not open up the endo-lysosomal vesicles and are probably released by localized destabilization of the endo-lysosomal membrane at the point of contact with NP, followed by extrusion of the NP through the membrane (Fig. 3G
). This hypothesis of NP escape from the endosomal compartment was further confirmed by the fact that polystyrene NPs, which did not show a charge reversal in the acidic pH as did PLGA NPs, were retained in the early and recycling endosomes or in the secondary endosomes, with most of the cytoplasmic compartment free of NP-associated green fluorescence (Fig. 4C
).
This mechanism of action of NPs is an important advantage in the use of PLGA NPs as cytoplasmic delivery vehicles. Unlike cationic lipids or polymers, PLGA NPs are cationic only in the endosomal compartment and do not destabilize the lysosomes. This reduces the chances of toxicity commonly associated with the use of cationic lipids and cationic polymers (33)
. Further, the intracellular uptake of the NPs is unaffected by serum (unpublished observation) and hence PLGA NPs are suitable for in vivo applications. NPs retained intracellularly could release the encapsulated drug slowly, leading to a sustained drug effect, which is especially crucial for drugs that require intracellular uptake. As a proof of this concept, we demonstrated a sustained and significantly greater antiproliferative efficacy of NP-encapsulated dexamethasone compared with dexamethasone in solution. Dexamethasone in solution showed inhibition while the cells were in contact with the drug (4 days). After the drug was removed (growth medium containing dexamethasone was replaced with fresh growth medium without any drug), the inhibitory effect of the drug was lost (Fig. 5A
). However, with NPs, a proportion of NPs would have entered the cytoplasm and remained there, slowly releasing the drug. This particular formulation of dexamethasone showed an approximate 70% drug release during 7 days under in vitro sink conditions (data not shown). Thus, despite removing NPs from the medium, cells would still have a continuous supply of the drug because of the NPs localized intracellularly. This result is reflected in the antiproliferative efficacy: the inhibition of cell proliferation in the dexamethasone NP group was significantly greater and sustained compared with the dexamethasone solution group (Fig. 5C
). Intracellular sustained release of the drug coupled with the fact that the receptors for dexamethasone are cytoplasmic could have resulted in the observed enhancement of NP-encapsulated dexamethasone. Results of these studies could explain the significant decrease in neointimal formation observed in our previous studies with localized delivery of dexamethasone-loaded NPs compared with the controls in a rat carotid model of restenosis (13)
.
In addition, we hypothesized that for NPs to function as a gene transfection system they should escape the endo-lysosomal compartment and slowly release the encapsulated DNA in the cytosol, resulting in sustained gene expression. To demonstrate this concept, we showed sustained gene expression of a marker gene in a prostate cancer cell line. The sustained gene expression observed in this study further substantiates the fact that NPs escape the endo-lysosomal compartment and that the DNA from the NPs is released slowly in the cytoplasmic compartment for their nuclear localization. We previously showed that encapsulated plasmid DNA is released at a sustained rate from PLGA nanoparticles (82% cumulative release during 17 days) under in vitro sink conditions (12)
. Transfection experiments with a commercially available transfecting agent, FuGene 6 (6:1 FuGene 6:DNA, 0.2 µg of DNA/well, Roche Diagnostics), resulted in a peak expression level at 2 days (250,000 pg/mg of cell protein), which then declined by about 70% in 3 days, suggesting that sustained gene expression observed with NPs could be due to the sustained intracellular release of DNA from NPs.
|
CONCLUSIONS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
|---|
|
ACKNOWLEDGMENTS |
|---|
We would like to thank Mr. Tom Bargar and Ms. Janice Taylor, of the electron and confocal laser microscopy core facilities at UNMC, for their assistance with the microscopic studies, and Ms. Elaine Payne for providing administrative assistance.
Received for publication February 15, 2002.
Revision received April 18, 2002.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONSREFERENCES |
|---|
This article has been cited by other articles:
|
|
 |
|
  M. Kubo, K. Egashira, T. Inoue, J.-i. Koga, S. Oda, L. Chen, K. Nakano, T. Matoba, Y. Kawashima, K. Hara, et al. Therapeutic Neovascularization by Nanotechnology-Mediated Cell-Selective Delivery of Pitavastatin Into the Vascular Endothelium Arterioscler. Thromb. Vasc. Biol., June 1, 2009; 29(6): 796 - 801. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Kimura, K. Egashira, L. Chen, K. Nakano, E. Iwata, M. Miyagawa, H. Tsujimoto, K. Hara, R. Morishita, K. Sueishi, et al. Nanoparticle-Mediated Delivery of Nuclear Factor {kappa}B Decoy Into Lungs Ameliorates Monocrotaline-Induced Pulmonary Arterial Hypertension Hypertension, May 1, 2009; 53(5): 877 - 883. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Nakano, K. Egashira, S. Masuda, K. Funakoshi, G. Zhao, S. Kimura, T. Matoba, K. Sueishi, Y. Endo, Y. Kawashima, et al. Formulation of Nanoparticle-Eluting Stents by a Cationic Electrodeposition Coating Technology: Efficient Nano-Drug Delivery via Bioabsorbable Polymeric Nanoparticle-Eluting Stents in Porcine Coronary Arteries J. Am. Coll. Cardiol. Intv., April 1, 2009; 2(4): 277 - 283. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. Hasadsri, J. Kreuter, H. Hattori, T. Iwasaki, and J. M. George Functional Protein Delivery into Neurons Using Polymeric Nanoparticles J. Biol. Chem., March 13, 2009; 284(11): 6972 - 6981. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Djordjevic, L. S. Del Rosario, Jinzhong Wang, and K. E. Uhrich Amphiphilic Scorpion-like Macromolecules as Micellar Nanocarriers Journal of Bioactive and Compatible Polymers, November 1, 2008; 23(6): 532 - 551. [Abstract] [PDF]
|
|
|
|
 |
|
 S. Kimura, K. Egashira, K. Nakano, E. Iwata, M. Miyagawa, H. Tsujimoto, K. Hara, Y. Kawashima, R. Tominaga, and K. Sunagawa Local Delivery of Imatinib Mesylate (STI571)-Incorporated Nanoparticle Ex Vivo Suppresses Vein Graft Neointima Formation Circulation, September 30, 2008; 118(14_suppl_1): S65 - S70. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Colognato, A. Bonelli, J. Ponti, M. Farina, E. Bergamaschi, E. Sabbioni, and L. Migliore Comparative genotoxicity of cobalt nanoparticles and ions on human peripheral leukocytes in vitro Mutagenesis, September 1, 2008; 23(5): 377 - 382. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 X.-P. Guan, D.-P. Quan, K.-R. Liao, Tao Wang, Peng Xiang, and K.-C. Mai Preparation and Characterization of Cationic Chitosan-modified Poly(D,L-lactide-co-glycolide) Copolymer Nanospheres as DNA Carriers J Biomater Appl, January 1, 2008; 22(4): 353 - 371. [Abstract] [PDF]
|
|
|
|
 |
|
 J. K. Vasir and V. Labhasetwar Preparation of Biodegradable Nanoparticles and Their Use in Transfection CSH Protocols, January 1, 2008; 2008(2): pdb.prot4888 - pdb.prot4888. [Abstract] [Full Text]
|
|
|
|
 |
|
 W. Lu, Q. Sun, J. Wan, Z. She, and X.-G. Jiang Cationic Albumin-Conjugated Pegylated Nanoparticles Allow Gene Delivery into Brain Tumors via Intravenous Administration Cancer Res., December 15, 2006; 66(24): 11878 - 11887. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. M. Moghimi, A. C. Hunter, and J. C. Murray Nanomedicine: current status and future prospects FASEB J, March 1, 2005; 19(3): 311 - 330. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Muro, X. Cui, C. Gajewski, J.-C. Murciano, V. R. Muzykantov, and M. Koval Slow intracellular trafficking of catalase nanoparticles targeted to ICAM-1 protects endothelial cells from oxidative stress Am J Physiol Cell Physiol, November 1, 2003; 285(5): C1339 - C1347. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
