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. Author manuscript; available in PMC: 2014 Feb 1.
Published in final edited form as: Osteoarthritis Cartilage. 2012 Nov 24;21(2):377–384. doi: 10.1016/j.joca.2012.11.011

Pharmaceutical Nanocarrier Association with Chondrocytes and Cartilage Explants: Influence of Surface Modification and Extracellular Matrix Depletion

KA Elsaid 1, L Ferreira 1, T Truong 1, A Liang 1, J Machan 2, GG D’Souza 1
PMCID: PMC3556184  NIHMSID: NIHMS424457  PMID: 23186944

Abstract

Objective

To evaluate cartilage diffusion and isolated chondrocyte association of micelles and liposomes and to determine the effect of cell-penetrating peptide (CPP) surface functionalization and extracellular matrix depletion on chondrocyte association and cartilage diffusion, respectively.

Methods

Rhodamine-labeled micelles and liposomes were incubated with bovine chondrocytes and cell-associated fluorescence was quantified using flow cytometry. Rhodamine-labeled CPP-modified micelles and liposomes were incubated with chondrocytes and cell-associated fluorescence was compared to unmodified nanocarriers. Rhodamine-labeled micelles and liposomes were incubated with bovine cartilage explants for 1, 2 and 4 hours and cartilage-associated fluorescence was compared across groups. Cartilage explants were treated with interleukin-1 alpha (IL-1α) or with 0.25% trypsin. Rhodamine-labeled micelles and liposomes were incubated with control, IL-1 and trypsin-treated explants and cartilage-associated fluorescence was compared across groups.

Results

Chondrocyte-associated fluorescence following treatment with micelles was significantly higher (P<0.001) than fluorescence in the cells treated with liposomes while there was no difference between cell-associated fluorescence in the liposomes-treated and untreated controls. CPP-modified nanocarriers exhibited a significant increase in chondrocyte association compared to unmodified nanocarriers (P<0.001). Micelles exhibited a time and concentration-dependent diffusion in cartilage explants while liposomes showed no diffusion. Following IL-1 and trypsin treatments, micelle diffusion in articular cartilage was significantly higher (P<0.001) than their diffusion in untreated explants.

Conclusion

Micelles exhibit superior association with isolated chondrocytes compared to liposomes. Surface modification with a CPP enhances chondrocyte association of both nanocarriers. 15 nm diameter Micelles are better than 138 nm diameter liposomes in penetrating articular cartilage and extracellular matrix depletion enhances micelle penetration.

Keywords: cell-penetrating peptide, cartilage, nanocarriers, osteoarthritis

Introduction

Owing to the involvement of a single or few joints, osteoarthritis (OA) is an ideal disease for localized drug delivery. The biggest challenge facing intra-articular (IA) drug administration in OA is the short residence time of drugs following IA injection. It is estimated that the joint fluid half-life of small drug molecules, e.g. non-steroidal anti-inflammatory drugs (NSAIDs) and large polymeric molecules e.g. hyaluronic acid and recombinant proteins are in the order of hours (1–5). Pharmaceutical nanocarriers e.g. micelles and liposomes are flexible platforms for target tissue specific delivery of drugs and imaging modalities (6–9). Given their potential, these nanocarriers could be used to enhance target joint tissue uptake of disease-modifying anti-osteoarthritis drugs (DMOADs).

The objectives of this study are to compare the time and concentration-dependent association of micelles and liposomes with articular chondrocytes and diffusion in cartilage explants, to examine the impact of early degenerative changes on their cartilage diffusivity and the impact of surface modification with a cell-penetrating peptide (CPP) on chondrocyte association. We hypothesized that micelles and liposomes would exhibit a time and concentration-dependent association with chondrocytes and cartilage tissue. Additionally, glycosaminoglycans (GAG) depletion would lead to an enhancement of nanocarrier cartilage diffusion and that nanocarrier surface modification with a CPP would result in enhanced chondrocyte association.

Methods

Rhodamine-labeled pharmaceutical nanocarrier preparation, characterization, and CPP modification

Liposomes and micelles were prepared by the thin film hydration method. The liposomal formulation consisted of phosphatidyl choline (PC), cholesterol, and rhodamine- phosphoatidylethanolamine (rhodamine-PE) in a molar ratio 69:30:0.27 while the micelle formulation consisted of methoxy (polyethylene glycol)-2000 (PEG-2K) and rhodamine- phosphoatidylethanolamine (rhodamine-PE) in a molar ratio of 99.5:0.5. Required amounts of lipids for each formulation were aliquoted from chloroform stock solutions and combined in a test tube. The choloroform was evaporated under reduced pressure at 40°C in a rotary evaporator to produce the dry lipid film that was then hydrated with an appropriate amount of phosphate buffered saline (pH 7.4). The resulting crude lipid suspension was subjected to probe sonication in 15–30 second pulses for no more than 5 minutes to produce the final preparations. The liposomes preparation had a final total phospholipid/cholesterol concentration of 5 mg/ml while the micelles had a final PEG-functionalized phosphatidyl ethanolamine concentration of 5 mg/ml. The fluorescence intensities of a 1:100 dilution of the micelles and liposomes at 5 mg/ml concentration were determined using a Hitachi Fluorospec 2000 spectrophotometer with an excitation/emission wavelengths of 575/595 nm and were compared to confirm that the preparations had equivalent fluorescence intensities. The HIV derived transactivator of transduction (TAT) peptide was used as a representative CPP for this study. To enable the preparation of CPP-modified carriers, DSPE-PEG 2K –TAT was synthesized using a slightly modified version of a previously described protocol (10). Briefly, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-2000] (ammonium salt) (DSPE-PEG(2000) maleimide) and TAT peptide (FITC-YGRKKRRQRRR-C) were dissolved separately in a buffer solution (50 mM triethanolamine hydrochloride, 50 mM sodium phosphate, 150 mM NaCl, and 1 mM EDTA, pH 8). An aliquot of TAT peptide solution containing 1mg of peptide was combined in an amber scintillation vial with sufficient DSPE-PEG (2000) maleimide stock solution to yield a mixture with a 2-fold molar excess of TAT over the DSPE-PEG (2000) maleimide. The mixture was stirred overnight at 4°C. The DSPE-PEG (2000)-TAT peptide solution was then dialyzed overnight against PBS at 4°C under constant stirring using Spectra/Pro dialysis membrane MWCO 6–8,000. Retention of FITC fluorescence after dialysis was used as a preliminary check that conjugation was successful followed by confirmation by TLC on silica plates using chloroform: methanol 80:20 v/v as mobile phase. Dry lipid films of the appropriate amounts of lipids were then hydrated with an appropriate amount of the aqueous DSPE-PEG (2000)-TAT peptide stock solution to yield either CPP-modified liposome or micelle preparations with 2.5 mg/ml total lipid and 2 mole % DSPE-PEG (2000)-TAT. Characterization of the different preparations was performed using dynamic light scattering.

Bovine articular cartilage explants harvesting and chondrocyte isolation

Full-thickness bovine cartilage explants were drilled from the medial and lateral sides of the femoral condyles of skeletally-mature young animals, obtained from a local slaughterhouse, with no gross signs of cartilage degeneration. The animals were 20–30 months of age. We have harvested a total of 155 explants from 18 animals for the purpose of our experiments. The two hind knee joints of each animal were used to harvest the explants at each harvest date. The explants yield varied from 6 to 13 per harvest. All explants were 12 mm in diameter and consistently less than 1 mm in thickness. Explants were washed repeatedly with sterile phosphate-buffered saline and cultured in DMEM supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin at 37°C and 5% CO2. Prior to conducting the tissue diffusion experiments, explants were embedded in 1% sterile agarose to prevent movement of the explants in the wells of the culture plates.

To isolate chondrocytes, cartilage slices from the tibial plateau and the femoral condyles were finely sliced and incubated with sterile 1 mg/ml collagenase from clostridium histolyticum (Sigma Aldrich, USA) in serum-free DMEM for 5 hours to overnight at 37°C with shaking. The cell suspension was then passed through a 70-μm cell strainer (BD Biosciences, USA) to remove tissue debris and chondrocytes were pelleted by centrifugation at 1,500 rpm for 5 min. The cell pellet was washed twice using DMEM+10% FBS and resuspended chondrocytes were seeded at 500,000 cells/well using 12-well culture plates (Costar, USA). Bovine chondrocytes were cultured in a monolayer and nanocarrier association experiments were performed within 7 days of plating the cells.

Incubation of unmodified and CPP-modified micelles and liposomes with bovine chondrocytes, flow cytometry and assessment of cytotoxicity

Rhodamine-labeled micelles and liposomes were incubated with bovine chondrocyte cultures at 0.5 mg/ml for 1, 2 and 4 hours at 37°C. Following incubation, cells were harvested from culture wells by trypsinization. Chondrocytes were incubated with 0.25% trypsin (500 μl per well for 10 min at 37°C). Trypsinization was terminated by adding 1 ml of DMEM+10%FBS to each well and 3 wells were pooled together, centrifuged at 1,500 rpm for 5 min and the cell pellet was washed with DMEM+10% FBS followed by PBS. Cells were fixed using 1% paraformaldehyde and cell-associated fluorescence was quantified using flow cytometry with 488 nm excitation laser with a 585/42 nm emission filter. In a separate set of experiments, CPP-modified or unmodified micelles and liposomes were incubated with bovine chondrocyte culture at 0.5 mg/ml for 1 hour and cell harvest and analysis was performed as described above. The reported mean values of chondrocyte-associated fluorescence intensities across treatments represent two independent experiments with chondrocytes isolated from cartilage tissue of a total of 4 unique animals with an n=3 (each “n” represents the pooling of 3 wells) for each treatment at each time point.

Assessment of the cytotoxic effect of unmodified or CPP-modified micelles and liposomes was performed by incubating the 4 preparations at 0.5 mg/ml with bovine chondrocytes (500,000 cells per ml; 100 μl per well) for 24 hours at 37°C in sterile 96-well plates. Cell viability was determined colorimetrically using the Aqueous One cell proliferation assay (Promega, USA). Data is presented as mean percentage cell viability ± standard deviation (SD) compared to untreated control. The data represent an average of two independent assays, each with 6 replicates per plate.

Interleukin-1 alpha (IL-1 α) and trypsin treatments of bovine cartilage explants

Bovine cartilage explants were incubated with recombinant human IL-1 α (R & D systems, USA) to a final concentration of 5 ng/ml in serum-free DMEM + 1% penicillin/streptomycin for 7 days with IL-1 supplementation and media changes every 2–3 days. The release of GAG from cartilage explants was determined using the dimethylmethylene blue (DMMB) dye binding assay as described (11). Histological evaluation of IL-1 stimulated explants was performed using alcian blue staining and was used to confirm GAG depletion from the extracellular matrix. Trypsin treatment was performed using 0.25% trypsin (3 ml per well) for 40 min at 37°C.

Incubation of unmodified micelles and liposomes with native, IL-1α and trypsin-treated cartilage explants

Studies of time-dependent association of rhodamine-labeled nanocarriers were performed using 0.5 mg/ml with 1, 2 and 4 hour incubations. Studies of concentration-dependent association of micelles with native articular cartilage explants were performed using 0.5, 0.28 and 0.0625 mg/ml with 1 hour incubation. Following IL-1α or trypsin treatment, rhodamine-labeled micelles and liposomes, both at 0.5 mg/ml concentration, were incubated with cartilage explants for 1 and 4 hours, respectively. Following incubations, explants were rinsed with DMEM to remove unbound nanocarriers and subsequently prepared for cryosectioning. Serial cartilage sections (10 μm) were obtained and imaged using a Nikon Eclipse E600 fluorescence microscope. Analysis of digital fluorescence micrographs was performed using Image J software. At least 3 sections were evaluated per cartilage explant and fluorescence intensities from a standard region of interest (ROI) were averaged across sections. Control cartilage explants were used to adjust for cartilage auto-fluorescence.

Statistical analyses

Absolute fluorescence intensities of nanocarrier association with articular cartilage were initially tested for equal variance and normality. The fluorescence intensities from different treatments were normally distributed, but had different variances. When applicable, mean values were reported with 95% confidence interval (CI). Chondrocyte association data is presented as mean geometric fluorescence ± SD. The 12 explants of any experimental group were derived from a range of 3 to 7 unique animals. A generalized estimating equation for Gaussian-distributed data in a cell means model was used to model fluorescence intensities as a function of experimental group while accounting for within-animal nesting of explants. Different cell types were permitted to have differing variances and further model misspecification was adjusted for using classical sandwich estimation. All hypotheses were tested using orthogonal linear estimates, with alpha maintained across all comparisons at 0.05 using the Holm-Shaffer step-down approach, with adjusted p-values used to draw inferences. This model was performed using SAS version 9.12. Chondrocyte association data were analyzed using t-test for normally-distributed or Whitney Rank-Sum for non-normally-distributed data for 2-group comparisons and analysis of variance (ANOVA) for normally-distributed or ANOVA on the ranks for non-normally distributed data for multiple group comparison. This analysis was performed using Sigma Plot, version 11.

Results

Size Distribution of unmodified and CPP-modified micelles and liposomes

The mean effective diameter of unmodified micelles was 15 nm with a polydispersity index of 0.4 compared to a mean effective diameter of 106 nm and a polydispersity index of 0.3 in the CPP-modified micelles. The mean effective diameter of unmodified liposomes was 138 nm and a polydispersity index of 0.2 compared to a mean effective diameter of 397 nm and a polydispersity index of 0.4 in the CPP-modified liposomes.

Association of micelles and liposomes with bovine chondrocytes and effect of CPP-modification

Association of rhodamine-labeled micelles and rhodamine-labeled liposomes with bovine chondrocytes is presented in figure 1A. Following 1, 2 and 4 hr incubations, chondrocytes treated with micelles showed significantly higher (P<0.001) mean geometric fluorescence compared to liposomes-treated and untreated chondrocytes. There were no significant differences in the geometric means of fluorescence between the liposomes-treated and untreated chondrocytes. There were no significant differences among geometric means of fluorescence in the 1, 2 and 4 hr micelles-treated chondrocytes. Similarly, there were no significant differences among geometric means of fluorescence in the 1, 2 and 4 hr liposomes-treated chondrocytes. The differential association abilities of micelles and liposomes with chondrocytes are shown qualitatively in figure 1B. Following treatment with 0.125 mg/ml rhodamine-labeled micelles, chondrocyte-associated fluorescence was higher compared to chondrocytes treated with 0.125 mg/ml rhodamine-labeled liposomes.

Fig. 1. Association of rhodamine-labeled micelles and liposomes with bovine chondrocytes.

Fig. 1

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1A Flow cytometry analysis of chondrocyte-associated fluorescence following incubation with micelles or liposomes at 0.5 mg/ml for 1, 2 and 4 hours at 37°C. Data represent geometric mean ± S.D.

*Indicates that fluorescence intensity in the 1-hour micelle-treated cells was significantly higher (P<0.001) than liposome-treated and control cells.

**Indicates that fluorescence intensity in the 2-hour micelle-treated cells was significantly higher (P<0.001) than liposome-treated and control cells.

***Indicates that fluorescence intensity in the 4-hour micelle-treated cells was significantly higher (P<0.001) than liposome-treated and control cells.

1B Representative micrographs of rhodamine-labeled carriers association with chondrocytes. Top row: untreated chondrocytes (control). Middle row: chondrocytes incubated with rhodamine-labeled liposomes at 0.125 mg/ml for 1 hour. Bottom row: chondrocytes incubated with rhodamine-labeled micelles at 0.125 mg/ml for 1 hour. Left column: DIC light micrographs visualizing cell morphology. Middle column: fluorescence micrographs visualizing Hoechst staining of cell nuclei. Right column: fluorescence micrographs visualizing cell-associated rhodamine fluorescence.

A representative fluorescence histogram of the effect of CPP modification on liposome association with chondrocytes is shown in figure 2A. The shift in the fluorescence histogram of CPP-modified liposomes compared to unmodified liposomes indicates an increase in chondrocyte association as a consequence of CPP modification. Similarly, a shift in the fluorescence histogram of CPP-modified micelles compared to unmodified micelles was also observed (figure 2B). The mean geometric fluorescence of CPP-modified liposomes was 81.0±24.0 compared to 10.5±2.5 in unmodified liposomes. The mean geometric fluorescence of CPP-modified micelles was 64.7±9.3 compared to 19.1±0.5 in unmodified micelles. Fold changes in geometric fluorescence of unmodified and CPP-modified micelles and liposomes-treated chondrocytes compared to untreated cells is presented in figure 2C. Fold change in chondrocyte-associated fluorescence following treatment with CPP-modified liposomes was significantly higher (P=0.002) than cell-associated fluorescence following unmodified liposomes treatment. Likewise, fold change in chondrocyte-associated fluorescence following treatment with CPP-modified micelles was significantly higher (P<0.001) than cell-associated fluorescence subsequent to treatment with unmodified micelles, CPP-modified liposomes as well as unmodified liposomes. Finally, the chondrocyte viability following treatment with unmodified micelles was 93.2 ±0.8% compared to 100.7±2.4% in CPP-modified micelles. The chondrocyte viability following treatment with unmodified liposomes was 99.6±3.0% compared to 97.0±2.0% in CPP-modified liposomes.

Fig. 2. Impact of cell-penetrating peptide (CPP) surface modification of rhodamine-labeled carriers on their ability to associate with bovine chondrocytes.

Fig. 2

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2A Representative flow cytometry histograms showing cell-associated fluorescence following treatment with unmodified liposomes (blue), CPP-modified liposomes (green) or untreated cells (red).

2B Representative flow cytometry histograms showing cell-associated fluorescence following treatment with unmodified micelles (blue), CPP-modified micelles (green) or untreated cells (red).

2C Fold change of geometric mean of cell-associated fluorescence following 1-hour treatment with 0.5 mg/ml of each of unmodified liposomes, CPP-modified liposomes, unmodified micelles and CPP-modified micelles compared to untreated controls. Data represent geometric mean ± S.D.

*Indicates that fold change in cell-associated fluorescence following treatment with CPP-modified liposomes was significantly higher (P=0.002) than cell-associated fluorescence with unmodified liposomes treatment.

**Indicates that fold change in cell-associated fluorescence following treatment with CPP-modified micelles was significantly higher (P<0.001) than cell-associated fluorescence of unmodified micelles, CPP-modified liposomes and unmodified liposomes.

Diffusion of micelles and liposomes in bovine cartilage explants

Association of rhodamine-labeled micelles and liposomes with native cartilage explants is presented in figure 3A (n=12 in each group at each time point). At 1 hour, micelles-treated explants exhibited significantly higher (P<0.001) fluorescence compared to liposomes-treated explants. The mean fluorescence intensity in the micelles group was 10.0; 95% CI (7.2–12.7) compared to mean fluorescence intensity in the liposomes group of 3.9; 95% CI (3.0–4.8). At 2 hours, micelles-treated explants exhibited significantly higher (P=0.004) fluorescence compared to liposomes-treated explants. The mean fluorescence intensity in the micelles group was 16.4; 95% CI (11.0–20.7) compared to a mean fluorescence intensity in the liposomes group of 3.0; 95% CI (1.6–4.4). At 4 hours, micelles-treated explants exhibited significantly higher (P<0.001) fluorescence compared to liposomes-treated explants. The mean fluorescence intensity in the micelles group was 16.4; 95% CI (12.2–20.7) compared to mean fluorescence intensity in the liposomes group of 4.5; 95% CI (3.4–5.7). The fluorescence intensity of the 4-hr micelle incubation was significantly higher (P=0.015) compared to the fluorescence intensity of the 1-hr micelle incubation. There was no significant difference between the fluorescence intensities of the 1 and 2-hr micelles treatments (P=0.362) or between the fluorescence intensities between the 2 and 4-hr treatments (P=0.838).

Fig. 3. Time and concentration-dependent association of rhodamine-labeled micelles and liposomes with bovine cartilage explants.

Fig. 3

Fig. 3

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3A Absolute fluorescence intensity following incubation with 0.5 mg/ml rhodamine-labeled micelles or 0.5 mg/ml rhodamine-labeled liposomes at 1, 2 and 4 hours (n=12 in each group at each time points). Data represent mean±SD.

*Indicates that cartilage-associated fluorescence following incubation with micelles was significantly higher (P<0.001) than fluorescence following incubation with liposomes at 1 hour.

**Indicates that cartilage-associated fluorescence following incubation with micelles was significantly higher (P=0.004) than fluorescence following incubation with liposomes at 2 hours.

*** Indicates that cartilage-associated fluorescence following incubation with micelles was significantly higher (P<0.001) than fluorescence following incubation with liposomes at 4 hours.

3B Concentration-dependent association of micelles with cartilage explants. Fluorescently-labeled micelles were incubated at 0.50, 0.28 and 0.0625 mg/ml (n=12 in each group) with cartilage explants for 1 hour at 37°C followed by quantitative estimation of cartilage-associated fluorescence. Fluorescence intensity in the 0.28 mg/ml and 0.0625 mg/ml groups were normalized to the 0.50 mg/ml concentration. Data represent mean± SD.

*Indicates that 0.0625 mg/ml concentration fluorescence in the superficial layer of articular cartilage was significantly lower (P=0.003) than 0.50 mg/ml concentration. There was no significant difference between the 0.50 mg/ml and 0.28 mg/ml concentrations.

3C Representative fluorescence micrographs of micelles and liposomes association with cartilage explants following 1, 2 and 4 hours. Scale bars represent 0.2 mm.

Fluorescence intensities in the 1-hr incubation with 0.28 mg/ml and 0.0625 mg/ml micelles were normalized to the mean fluorescence intensity in the 1 hr incubation with 0.5 mg/ml micelles (figure 3B) (n=12 in each group). Compared to the 0.5 mg/ml concentration, the 0.28 mg/ml concentration normalized fluorescence was 77.8%; 95% CI (57.6–97.9), while the 0.0625 mg/ml concentration normalized fluorescence was 57.3%; 95% CI (51.7–62.9). The cartilage-associated fluorescence in the 0.0625 mg/ml group was significantly lower (P=0.003) than the cartilage-associated fluorescence in the 0.5 mg/ml with no significant difference between the 0.5 mg/ml and 0.28 mg/ml treatments and no significant difference between the 0.28 and 0.0625 mg/ml treatments.

Representative histological images of the 1, 2 and 4 hour incubations with rhodamine-labeled micelles and liposomes are presented in figure 3C. At 2 and 4 hours, micelles appeared to have diffused across the superficial and middle zones of articular cartilage. In contrast, rhodamine-labeled liposome diffusion into the articular cartilage was not observed.

Impact of IL-1 and trypsin treatments on diffusion of micelles and liposomes in cartilage explants

Stimulation with IL-1α has resulted in GAG depletion of cartilage explants as evidenced by a significant increase in GAG release from cartilage explants into the culture media (figure 4B). Representative alcian blue stained cartilage tissue demonstrated lack of staining in the IL-1 treated explants. In contrast, native cartilage explants demonstrated intense GAG staining (figure 4B). Fluorescence intensities of micelles and liposomes association with cartilage explants following IL-1 or trypsin treatments were normalized to control and are presented in figures 4A and 4C, respectively. Compared to control, and following IL-1 treatment, normalized mean fluorescence intensity with micelles treatment was 429.4%; 95% CI (349.0–509.8) while normalized mean fluorescence with liposomes was 132.0%, 95% CI (100.7–163.3). Normalized mean fluorescence with micelles in IL-1 treated explants was significantly higher (P<0.001) than mean fluorescence with micelles in control explants. There was no significant difference between mean fluorescence in control or IL-1 treated explants that were incubated with liposomes. Following trypsin treatment, normalized mean fluorescence intensity with micelles treatment was 284.4%; 95% CI (227.8–341.0) while normalized mean fluorescence with liposomes was 134.3%; 95% CI (105.0–163.7). Normalized mean fluorescence with micelles in trypsin-treated explants was significantly higher (P<0.001) than mean fluorescence with micelles in control explants. There was no significant difference between mean fluorescence in control or trypsin-treated explants that were incubated with liposomes. Representative fluorescence micrographs of control, IL-1 and trypsin treated explants that were incubated with rhodamine-labeled micelles or liposomes are presented in figure 4D. Compared to control, IL-1 and trypsin treatments have resulted in improved cartilage diffusion of micelles following 1 hour incubation. On the contrary, neither treatment has impacted the migration of rhodamine-labeled liposomes in the articular cartilage even with 4 hours of incubation.

Fig. 4. Impact of interleukin-1 alpha and trypsin treatments on rhodamine-labeled micelles and liposomes’ association with cartilage explants.

Fig. 4

Fig. 4

Fig. 4

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4A Normalized fluorescence intensity in control and IL-1 stimulated bovine cartilage explants. Treatment with rhodamine-labeled micelles was performed using 0.5 mg/ml micelles and incubation for 1 hour at 37°C. Treatment with rhodamine-labeled liposomes was performed using 0.5 mg/ml liposomes and incubation for 4 hour at 37°C. Data represent mean± SD.

*Indicates that fluorescence intensity in IL-1 treated explants incubated with rhodamine-labeled micelles (n=12) was significantly higher (P<0.001) than micelles-treated control explants (n=12).

4B 7-day cumulative glycosaminoglycans (GAG) release of IL-1-stimulated and control bovine cartilage explants (n=12 in each group). *Indicates that cumulative GAG release was significantly higher (p<0.001) in the IL-1 group compared to control. a) Representative alcian blue stained control bovine cartilage explants; b) Representative alcian blue stained IL-1-stimulated bovine cartilage explants. Scale bars represent 0.2 mm.

4C Impact of trypsin treatment on rhodamine-labeled micelle and liposome association with cartilage explants. Treatment with rhodamine-labeled micelles was performed using 0.5 mg/ml micelles and incubation for 1 hour at 37°C. Treatment with rhodamine-labeled liposomes was performed using 0.5 mg/ml liposomes and incubation for 4 hour at 37°C. Data represent mean± SD.

*Indicates that fluorescence intensity in trypsin-treated explants incubated with rhodamine-labeled micelles (n=12) was significantly higher (P<0.001) than micelles-treated control explants (n=12).

4D Representative fluorescence micrographs of micelles and liposomes association with cartilage explants in untreated (control) explants, IL-1 treated and trypsin-treated explants. Scale bars represent 0.2 mm.

Discussion

OA is a degenerative disease characterized by pathologic changes to cartilage, synovium and subchondral bone (12). A number of potential disease targets, suitable for pharmacologic interventions, have been identified (13–15). A significant proportion of these targets reside intracellularly in chondrocytes or in the extracellular matrix of cartilage. To successfully inhibit these disease pathways of interest, DMOADs have to maintain effective synovial fluid concentration, diffuse through the different layers of cartilage and distribute intraceullarly into chondrocytes. Optimizing the rate and extent of these kinetic processes (diffusion into cartilage and cellular uptake) can significantly improve the efficacy of these DMOADs.

We have evaluated the ability of micelles and liposomes (inert, biocompatible and widely-used drug carriers) to associate with chondrocytes. Micelles have shown an ability to associate with chondrocytes compared to liposomes which did not show any appreciable association. The cellular association of micelles was rapid as seen at 1 hour post-incubation with no significant change to the extent of this association at later time points. We have also studied the impact of surface functionalization of micelles and liposomes with a CPP on their chondrocyte association. Trans-activating transcriptional activator (Tat) protein facilitates translocation of the human immunodeficiency virus (HIV-1) across cell membrane (16, 17). TAT peptide is an arginin-erich, positively-charged peptide derived from Tat protein and is the most-widely used CPP (18, 19). TAT-conjugation was shown to efficiently improve cellular uptake of a variety of nanoparticles, e.g. micelles, liposomes, dendrimers, gold and silver (20–24). CPP conjugation of liposomes and micelles improved their chondrocyte association. The magnitude of improvement was higher with liposomes with an estimated 8-fold improvement in cellular association compared to unmodified liposomes. On the other hand, the magnitude of enhancement in chondrocyte association of micelles following CPP conjugation was approximately 3-fold. Nonetheless, CPP-modified micelles exhibited the most significant chondrocyte association amongst the four formulations. TAT-conjugation to micelles or liposomes did not adversely affect chondrocyte viability, an effect that is consistent with other observations documenting the lack of cytotoxic effect by the TAT peptide.

Cartilage is a dense avascular tissue and the rate of diffusion of solutes and particles in cartilage is highly variable and depends on factors e.g. particle size, concentration and extracellular matrix composition (25–27). In the bovine cartilage explant model, micelles exhibited a time-dependent diffusion in articular cartilage. In contrast, liposomes have shown no diffusion in articular cartilage. This difference in diffusivity was observed as early as 1 hour post incubation and remained in the 4-hr treatment. The one-week IL-1 α treatment was used to simulate early cartilage OA changes. This is characterized by GAG depletion with no significant degenerative change to the collagen network of the tissue (28). We expected that with GAG depletion, diffusion of micelles and liposomes will be enhanced. Interestingly, only micelles exhibited a significant enhancement in their cartilage penetration. There was no improvement in cartilage diffusion with liposomes following GAG depletion. Subsequent to trypsin treatment, diffusion of micelles in cartilage was also enhanced. Diffusion of liposomes into articular cartilage was not impacted by trypsin treatment. Taken together, these results point to the ability of micelles to diffuse into articular cartilage and that this diffusion is enhanced by early degenerative changes to articular cartilage.

The micelle and liposome formulations used in this study have been well characterized over more than a decade of study in the field of pharmaceutical nanocarriers. The protocol reproducibly yielded micelles of 15 nm effective diameter and liposomes of 138 nm effective diameter that are in agreement with literature-reported values. The CPP-modification resulted in a significant increase in the effective diameters of both carriers. The size difference between unmodified micelles and liposomes can be used to partially explain differences seen in cartilage diffusivity. However, size difference may not solely explain differential cartilage diffusion. Surface characteristics may play a role in the interaction of micelles and liposomes with cartilage. In this study, micelles had a surface composed of polyethylene glycol that is more hydrophilic than liposomes’ surface. It was evident that liposomes were slower to associate with the top most layer of cartilage compared to micelles. With 4 hours of incubation, the fluorescence intensity of liposomes-associated cartilage remained lower than the fluorescence intensity of the 1-hr micelles-associated cartilage. The hydrophilic nature of lubricin, the large glycoprotein bound to the surface of articular cartilage (29), may explain this disparity through favorable interaction with the more hydrophilic micelles. Conversely, the phospholipids from the liposomal bilayer may associate with the surface-associated phospholipids (SAPLs) (30) in the superficial layer of articular cartilage, precluding further diffusion. Thermodynamically, the PEG-PE monomers may prefer the micellar conformation and hence continue to diffuse across the superficial zone into the middle zone of the cartilage. GAG depletion significantly enhanced diffusion of micelles into the middle zone of articular cartilage. This is expected as removal of GAG, a major extracellular matrix component, reduces steric hindrance to particle movement. GAG removal did not significantly enhance diffusion of liposomes, providing more evidence that the barrier to diffusion of liposomes into articular cartilage may be the nature of the top-most superficial layer of articular cartilage.

The difference in association of unmodified micelles and liposomes with chondrocytes may be partially explained by their size differences. Smaller size particles will probably better interact and associate with chondrocytes’ cell membrane (31). While CPP-modification enhanced chondrocyte association of both carrier systems, a greater improvement was seen with liposomes. One potential explanation of this difference is the number of CPP-residues per particle. The larger-surface liposomes would allow a greater number of CPP-residues per particle compared to micelles. A range of 1–4 CPP residues per particle is sufficient to confer cell penetrating properties, as previously reported (19). We, however, did not determine the specific number of residues per particle as we had reasonable assurance that the molar ratio of our conjugation procedure more than satisfies this threshold.

We have not evaluated the impact of CPP-surface modification and the resultant increase in effective particle diameters on cartilage explant diffusion of micelles. Therefore the effect of particle size variation in the same nanocarrier platform on cartilage penetration has not been assessed in this study. Another limitation to this work is the in-vitro nature of the experiments. Whether the observed differences in chondrocyte association and cartilage penetration between micelles and liposomes in the explant model translates to in-vivo differences following IA injection remains to be studied. Previous in-vivo experiments performed with liposomes in rheumatoid arthritis (RA) animal models suggest that liposomes are predominantly uptaken by the inflamed synovium (32, 33). While this is clinically valuable in RA, our work suggests that liposomes may not be a suitable drug carrier platform to target articular cartilage and thus of limited value in OA. The novelty of our work lies in the use of a widely-established explant model coupled with isolated chondrocyte culture to study their interaction with different pharmaceutical platforms. This model can be used to understand the effect of surface modification, size and surface charge of various nanocarriers on diffusion in, and association with articular cartilage. Using the explants model, we were able to demonstrate that micelles, either modified or unmodified, appear to be a suitable drug carrier platform to deliver cartilage-targeted intracellular poorly soluble drugs and thus warrant their further development as a drug delivery platform for OA.

Acknowledgments

This work is supported by NIH/NIAMS R15 AR061722-01 to KE and GD.

Role of Funding Source: Funding source had no role in the design, collection, and interpretation of the data or the decision to submit for publication.

Footnotes

Author Contributions: All authors were involved in drafting the article or revising it critically for content, and all authors approved the final version to be published. Dr. Elsaid and Dr. D’Souza had full access to all of the data in the study and take responsibility for the data integrity and data analysis accuracy.

Study conception and design: Elsaid, D’Souza

Data acquisition: Elsaid, Ferreira, Truong, Liang, D’Souza

Data analysis and interpretation: Elsaid, Ferreira, Truong, Machan, D’Souza

Conflict of Interest Statement: All authors have no potential conflict to declare related to the content of the manuscript.

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