Cartilage Penetrating Nanocarriers Improve Delivery and Efficacy of Growth Factor Treatment of Osteoarthritis

Abstract

Osteoarthritis is a debilitating joint disease that affects over 30 million people and has no disease-modifying therapies. Disease-modifying osteoarthritis drugs have been tested in the clinic, but all have been unsuccessful in clinical trials. A key point of failure for several of these drugs has been inefficient and inadequate delivery to target cells. Anabolic growth factors are one class of such drugs which could be disease-modifying if successfully delivered directly to chondrocytes, which reside deep within dense, anionic cartilage tissue. To overcome this biological barrier, we conjugated a growth factor to a small, cationic nanocarrier for sustained, targeted delivery to chondrocytes following direct intra-articular injection. The nanocarrier utilizes reversible electrostatic interactions with anionic cartilage tissue to improve tissue binding, penetration, and residence time. Amine terminal polyamidoamine (PAMAM) dendrimers were end-functionalized with variable molar ratios of PEG to control surface charge. From this small family of variably PEGylated dendrimers, an optimal formulation was selected, with 70% uptake into cartilage tissue and 100% cell viability. When conjugated to insulin like growth factor 1 (IGF-1), the dendrimer penetrated bovine cartilage of human thickness within 2 days and enhanced therapeutic IGF-1 joint residence time in rat knees by 10-fold, for up to 30 days. In a surgical model of rat osteoarthritis, a single injection of dendrimer-IGF-1 rescued cartilage and bone more effectively than free IGF-1. Dendrimer-IGF-1 reduced width of cartilage degeneration by 60% and volumetric osteophyte burden by 80% relative to untreated rats at 4 weeks post-surgery. These results suggest that PEGylated PAMAM dendrimer nanocarriers could improve pharmacokinetics and efficacy of disease modifying osteoarthritis drugs in the clinic.

One Sentence Summary:

A charged molecular carrier enables delivery of a growth factor to its target cells within cartilage over an extended time, improving delivery of the growth factor with therapeutic benefit to cartilage, bone, and synovium in a rat model of osteoarthritis.

Introduction

Osteoarthritis (OA) is a debilitating disease of individual joints which manifests as degeneration of articular cartilage, causing serious pain and impeding mobility. It affects 20-30 million people in the U.S. alone with an estimated economic impact of $60 billion/year (1, 2). Despite this enormous unmet medical need, no disease-modifying drug exists, and the current standard of care is limited to palliative treatment to manage symptoms as the disease progresses. While several classes of drugs have shown promise in preclinical studies, including anti-inflammatory small molecules, cytokine receptor antagonists, anabolic growth factors, and targeted inhibitors of catabolic enzymes, all have failed in clinical development (3). Even the current pharmacological standard of care for pain, specifically intra-articular corticosteroids and hyaluronic acid, is subject to intense debate on safety and efficacy (4, 5). Many of these shortcomings are rooted in inadequate drug delivery (6, 7).

Even when directly injected into the affected joint, the rapid clearance rate of molecules from the joint space and dense, avascular cartilage tissue constitute two considerable biological barriers to drug delivery to chondrocytes. Many compounds of interest have intra-articular half-lives as short as 2-4 hours (7, 8). Combined with a need for clinicians to minimize repeat intra-articular injections, the short half-life of these drugs limits their duration of therapy and thus limits overall efficacy. In a retrospective clinical analysis (NCT00110916), Chevalier et al. identified insufficient drug delivery as a key factor in the Phase II OA failure of an interleukin 1 receptor antagonist (IL1-RA, Anakinra), an approved disease-modifying rheumatoid arthritis drug. Patients received a single injection of IL1-RA over a 12 week study, yet the drug was below its limit of quantification in serum after 24 hours (9).

Previous studies within our group and others have shown that small (< 15 nm) cationic nanocarriers can overcome the biological barriers of the joint by binding and penetrating anionic cartilage tissue faster than the carriers can be cleared from the joint space (10-16). It is vital for clinical translation of these cartilage penetrating nanocarriers to identify an optimal nanocarrier surface charge that is both safe and efficacious. Our goal in this study was to engineer a family of cartilage penetrating nanocarriers with variable surface charge, select an optimally charged system, and rigorously test that system for improved drug delivery and efficacy in both in vitro and in vivo models.

Beyond small size and tunable surface charge, other important considerations for a translatable cartilage penetrating nanocarrier include scalable synthesis, robust characterization, and flexibility in accommodating different classes of therapeutic cargo. We found that polyamidoamine (PAMAM) dendrimers, generations 4-6, fit all design criteria well. These hierarchically branched macromolecules are <10 nm in diameter with a dense surface functionality of 64-256 cationic primary amines. These amines can be modified with poly(ethylene glycol) (PEG) oligomers at nearly stoichiometric conversions for tight control of charge and improved biocompatibility (17-20). The surface amines also provide a versatile chemical handle for conjugation to a variety of drugs (19, 21). PAMAM dendrimers are produced commercially at large scale and can be thoroughly characterized by well-established methods (18, 22, 23).

We chose to use insulin-like growth factor 1 (IGF-1) as our therapeutic cargo. IGF-1 is an anabolic growth factor that promotes chondrocyte survival, proliferation, and biosynthesis of cartilage matrix macromolecules (24-26). It also shows anti-inflammatory effects in cytokine challenged cartilage tissue (24, 27). For these properties, IGF-1 has garnered significant interest as a potential disease-modifying osteoarthritis drug.

Here, we identified an optimally charged PEGylated dendrimer capable of high uptake into cartilage tissue without toxicity and conjugated it to insulin like growth factor 1 (IGF-1) without loss in bioactivity. The dendrimer-IGF-1 conjugates penetrated full thickness cartilage (~1 mm) mimicking that of a human and maintained therapeutically relevant concentrations in the joints of rats for 30 days following intra-articular injection. In a well-characterized rat surgical model of osteoarthritis (28-31), a single intra-articular injection of dendrimer-IGF-1 provided a significant reduction of cell and aggrecan loss relative to untreated rats at 4 weeks post-surgery, whereas free-IGF-1 did not. Both free IGF-1 and dendrimer-IGF-1 reduced osteophyte formation in the injured joints, but this effect was only statistically significant relative to untreated rats for dendrimer-IGF-1. This study provides evidence that conjugation to a cartilage penetrating PEGylated dendrimer can improve both delivery and efficacy of disease-modifying biologic drugs in osteoarthritis.

Results

Dendrimer-IGF-1 conjugation retains IGF-1 bioactivity

PAMAM dendrimers are densely cationic macromolecules, and thus have the capability to bind to anionic cartilage tissue. We hypothesized that by binding to cartilage, they could avoid rapid clearance of the synovial fluid through joint microvasculature (Figure 1A). Their small size could enable them to penetrate dense cartilage tissue to access IGF-1 receptors on chondrocytes throughout the entirety of the tissue; the presence of partial PEG shielding of surface charge could enable dynamic binding-unbinding interactions that would favor deep penetration into cartilage over surface binding. Generation 4 (14 kDa, 64 NH₂ end groups) and Generation 6 (58 kDa, 256 NH₂ end groups) dendrimers were used (Figure 1B, Gen 4 shown) and 0-60% of end groups were PEGylated to covalently and sterically shield surface charge, creating a panel of variably charged formulations for testing in model systems. Remaining unreacted primary amine end groups contribute to positive surface charge.

Figure 1. Design and synthesis of bioactive PEGylated dendrimer – IGF-1 conjugates as cartilage penetrating nanocarriers.

(A) Schematic of drug and carrier fates within joints following intra-articular injection based on size and charge. Material within the synovial fluid is rapidly cleared from the joint. Cationic material binds to anionic aggrecan within cartilage to avoid clearance. Small cationic materials can penetrate the aggrecan matrix to interact with chondrocytes deep in the tissue. (B) Chemical structure and key design characteristics of PAMAM dendrimers (Gen 4 shown) as cartilage penetrating nanocarriers. (C) Synthetic scheme of a partially PEGylated dendrimer – IGF-1 formulation. For the Gen 4 35% PEG formulation, n = 64, × ~= 18, y ~= 4.4, z ~= 0.6. For the Gen 6 45% PEG formulation, n = 224, x~= 90, y~= 9.4, z~= 0.6. IGF1 color code: orange, IGF-1 receptor binding site (PheTyrPhe); blue, lysine (alternative reaction site); cyan, matrix IGF-1 binding protein site (hinders IGF-1 transport); magenta, fluorescent tracer. (D) Rate of biosynthetic ³⁵S sulfate incorporation from media into ex vivo bovine cartilage following treatment with no IGF-1, dendrimer-IGF-1, or free IGF-1. Data are means + 95% CI, N = 10 explants, statistics by one way ANOVA with Tukey post-hoc tests. (E) Percent of NIH 3T3 cells in S phase following 24 hours of treatment with no IGF-1, dendrimer-IGF-1, or free IGF-1, as determined by cell cycle flow cytometry with DAPI and EdU staining. Data are means + 95% CI, N = 6 biological replicates, statistics by one way ANOVA with Tukey post-hoc test. (F) Confocal image of interaction of dendrimer-IGF-1 conjugates with live bovine cartilage tissue. Binding to extracellular matrix and dense binding to cellular membrane (white arrows) are visible.

The dendrimer-IGF-1 bioconjugation scheme is shown in Figure 1C, resulting in a nondegradable thiol-maleimide linkage. The dendrimer-IGF-1 conjugate retained equivalent bioactivity to free IGF-1, both inducing sulfated proteoglycan synthesis in bovine cartilage explants (Figure 1D) as determined by ³⁵S sulfate incorporation and inducing proliferation in NIH 3T3 fibroblasts as determined by DAPI+EdU cell cycle analysis in flow cytometry (Figure 1E, S1) on par with free IGF-1. Confocal microscopy of sections of bovine cartilage tissue explants incubated with fluorescent dendrimer-IGF-1 revealed colocalization of dendrimer-IGF-1 with cartilage tissue and cellular membranes (Fig. 1F). Based on the combined evidence of Figs. 1D-F, the site of membrane localization is likely the extracellular IGF-1 receptor, though some nonspecific electrostatic interaction with the membrane is also possible.

Screening identifies optimally PEGylated dendrimers

PEGylation of the panel of Gen 4 and 6 dendrimers was quantified by ¹H NMR and MALDI-TOF mass spectrometry as a percentage of n total end groups (Figures 2A, S2, S3, and Tables S1, S2). Dendrimer formulations are hereafter referred to as Gen A – B%, where A is dendrimer generation and B is the % of terminal amines PEGylated with PEG (MW = 436 Da, x_avg = 8). The panel of partially PEGylated dendrimers were then incubated with bovine cartilage tissue disks for 24 hours (Figure 2B, 2C). Cartilage uptake was measured as the percentage of dendrimer from a 300 μL bath within a ~ 7 μL explant (Figure 2D, colored and S4). The PEGylated dendrimers were counter-screened for cell viability of human CHON-001 cells after treatment for 48 hours (Figures 2D, black and S5). Dendrimer formulations with higher % PEGylation (less charge) showed reduced uptake into anionic cartilage tissue, but also greater cell viability. All formulations showed some preferential partitioning into cartilage greater than equilibrium diffusion into the cartilage as indicated by uptake > 2.3 %. When cartilage with bound dendrimers was transferred to 10x PBS, nearly 100% of dendrimers desorbed from the tissue, further suggesting an electrostatic dendrimer-cartilage binding mechanism that can be shielded in the presence of free ions in the hyperosmotic PBS (Figure S6). Taken altogether, these data were used to select one formulation from each generation for further testing; the selected systems show the highest % uptake possible while maintaining 100% cell viability at 10 μM dendrimer (Figure 2D). Gen 4 35% PEG (42 free surface amines) and Gen 6 45% PEG (123 free surface amines) were selected for further study, with mean uptakes of 51% and 71%, respectively, and ~100% viability.

Figure 2. Screen identifies optimal degree of dendrimer PEGylation for enhanced electrostatic cartilage binding without toxicity.

(A) Synthesis (left) and characterization (right) of dendrimers with varying lengths and molar ratios of PEG grafted to end groups. (B) Schematic of uptake and viability experiments involving bovine cartilage tissue explants. (C) Bright field image of cartilage following uptake. (D) 24 hour tissue uptake and 48 hour cell viability after treatment with 10 µM partially PEGylated dendrimers (PEG MW: 436 Da, x_avg = 8). Theoretical surface charge shown. Data are means + 95% CI, N = 15 explants for uptake, N = 4 technical replicates for viability, *** P<.001 vs. all others by one way ANOVA & Tukey HSD post-tests. (E) Cell viability staining of sectioned bovine cartilage explants following 48 hours of incubation with PEGylated dendrimers. Cell death at edges is due to artifact from tissue harvest. Scale bar 200 μm. (F) Histology (H&E) of rat synovium and cartilage following intra-articular injection of nanocarrier. Scale: 200 µm. B: Bone, C: Cartilage

Optimally PEGylated dendrimers do not exhibit acute or chronic histotoxicity

The two formulations selected from screening were further tested for toxicity in ex vivo bovine cartilage tissue disks and in vivo in rats. PEGylated dendrimers were incubated at 1 and 10 μM with cartilage disks for 48 hours, then tissue sections were cut, stained for live/dead cells, and imaged (Figure 2E, quantified in Figure S7). All dendrimer treated tissues showed no greater toxicity than the vehicle control – cell death at upper and lower edges is artifact from tissue harvesting. Healthy rats were then injected intra-articularly with dendrimer-IGF-1 to an approximate knee concentration of 10 µM dendrimer. Serum samples were taken from rats 2 and 7 days post-injection and levels of common toxicological biomarkers were analyzed (Table S3). All biomarker levels were either within reported normal ranges or statistically equivalent to uninjected animals. A second group of rats were sacrificed two months after injection for histological analysis of chronic organ damage. H&E stained sections of joints (Figure 2F), liver, kidney, and lungs (Figure S8) were found to be normal by an independent pathologist. We also tested an under-PEGylated dendrimer, Gen 4 20% PEG, that had shown mild cytotoxicity in our human chondrocyte cell line (Figure 2D). Gen 4 20% PEG induced considerable inflammation in the synovium of the rat knee, indicating the importance of optimized surface charge (Figure S9).

Dendrimer-IGF-1 considerably extends joint residence time of drug

Fluorescently labeled Gen 4 35% - IGF-1, Gen 6 45% - IGF-1, and free IGF-1 were injected intra-articularly (Figure 3A) into rat knees. Concentrations were tuned to ensure equal quantity of fluorophore in each formulation, and all formulations were injected to approximately 6 μM IGF-1 (~10 μM dendrimer) in the synovial fluid. IGF-1 fluorescence within joints was serially measured over a period of 1 month using an in vivo imaging system (IVIS, Figure 3B). Total radiant efficiency data within the anatomical region of interest were quantified and plotted over time (Figure 3C). Based on fitting to a single phase exponential decay function, IGF-1 had a joint half-life of only 0.41 days [95% CI 0.31 – 0.57 days], while Gen 4 35% extended that half-life to 1.1 days [0.84 – 1.5 days], and Gen 6 45% extended joint half-life even longer to 4.2 days [2.8 – 8.5 days] (Figure 3C). These half-lives and 95% CIs were used to calculate time at therapeutically relevant dose in the joint based on the initial injected joint concentration of 6 μM IGF-1 and aggrecan biosynthesis saturating IGF-1 concentration of 0.04 μM (24, 32) (Figure 3D). A single injection of Gen 6 45% PEG – IGF-1 provided 30.4 days at IGF-1 saturating conditions within the joint, relative to 7.8 days for Gen 4 35% PEG – IGF-1 and 2.9 days for free IGF-1.

Figure 3. PEGylated dendrimer nanocarriers extend joint residence, therapeutic time, and cartilage penetration of IGF-1 in rats.

(A) Schematic of rat intra-articular injection & IVIS image of uninjected rat to establish background signal. The field of view for panel B is shown in the red box (B) Representative in vivo imaging system (IVIS) images of rat joints over 28 days after injection of fluorescent IGF-1 formulations. Black circles represent the anatomical joint ROI used for quantification. Fluorescence scale: Max = 9.0e7, Min = 2.0e7 radiant efficiency unit (shown in SI in Equation 1). (C) Timecourse of fluorescent radiant efficiency within joints. Data are fit to a one phase exponential decay with a common plateau based on background signal. Data are means + 95% CIs of nonlinear fit, N = 8 joints per formulation. Half-lives were statistically different for each data set (P<0.0001) by extra sum of squares F test. (D) Estimated time at IGF-1 concentrations that saturate aggrecan biosynthesis activity for each delivery method, based on initial concentration after injection of ~6 µM IGF-1, saturating concentration of 0.04 µM, and mean + 95% CI of carrier half-life. (E) 3D reconstruction of multiphoton microscopy images of full thickness cartilage from intact rat femurs harvested 6 days post-injection.

Dendrimer – IGF-1 penetrates the full thickness of cartilage

Rats injected intra-articularly with fluorescent IGF-1 and dendrimer IGF-1 formulations were sacrificed at 2 and 6 days post injection. Joints were excised, split into tibia and femur, stained with a fluorescent aggrecan antibody, and imaged in a multiphoton confocal microscope throughout the depth of the cartilage tissue (120-160 μm), as indicated by aggrecan fluorescence (red) and second harmonic generation signal of collagen II (grey). Image stacks from the medial femoral condyle at 6 days post injection were reconstructed into 3 dimensions in Figure 3E. IGF-1 fluorescent signal (blue) is visible throughout the width and depth of the femoral condyle for both dendrimer-IGF-1 formulations, but strikingly absent in the case of free IGF-1. At 2 days post-injection (Figure S10), IGF-1 was still present within the femoral cartilage. A related experiment was conducted ex vivo in 1 mm thick bovine cartilage tissue disks (Figure 4A), which more accurately represent human cartilage in structure and thickness (1-2 mm) than rat cartilage. Explants were incubated for 2 or 6 days in 10 μM dendrimer-IGF-1, then sectioned and visualized across the direction of diffusion under confocal microscopy, as shown in Figure 4B. The quantified intensity profiles of these images are shown in Figure 4C. Greater area under the intensity profile indicates more uptake of the formulation into the entirety of the cartilage and an even distribution of intensity from right to left indicates more thorough diffusive penetration of the tissue. Even without any clearance mechanism in this experiment, free IGF-1 showed relatively little uptake and penetration of cartilage tissue relative to the dendrimer-IGF-1 formulations at both timepoints. After 2 days of uptake, the less charged Gen 4 35% PEG – IGF-1 formulation had diffused evenly throughout the tissue while the more charged Gen 6 45% PEG – IGF-1 still exhibited a concentration gradient across the cartilage thickness. After 6 days of uptake, Gen 6 45% achieved a uniform concentration profile across the cartilage at nearly twice the average fluorescence intensity of Gen 4 35%. In summary, the more charged Gen 6 45% took 6 days to achieve a uniform distribution throughout the tissue, but by that time, much more total dendrimer formulation had been taken up from the bath than the Gen 4 35%. The transport differences between the two formulations were more prominent in media containing no serum (Figure S11), in which electrostatic effects are inherently more pronounced.

Figure 4. Dendrimer-IGF conjugates exhibit full penetration of human thickness cartilage within window of residence time in joint.

(A) Schematic of live bovine cartilage explant harvest and penetration assay. Fluorescent dendrimer distribution in cartilage after 2 or 6 days in DMEM + 10% FBS + cartilage media supplements. Red box indicates field of view (FOV). (B) Confocal microscope images of fluorescently labeled IGF-1 (purple) across the diffusion gradient (right to left, -y) of the tissue (shown in bright field). Arrow indicates direction of dendrimer transport. Scale: 200 μm (C) Quantification of IGF-1 fluorescence intensity across the explant section. Average fluorescence intensity over the entire tissue section is shown. All images were taken under the same laser power, intensity, and offset.

Dendrimer – IGF-1 rescues cartilage from degeneration in a surgical rat model

Based on the results in Figures 3 and 4, Gen 6 45% - IGF-1 provides a higher dose of IGF-1 throughout the entirety of cartilage for a longer period of time than Gen 4 35% - IGF-1, and thus Gen 6 45% - IGF-1 was selected for testing in a rodent osteoarthritis model. Rats were injured in their right hind limb knee by Anterior Cruciate Ligament Transection + Partial Medial Meniscectomy (ACLT+MMx), which is a well characterized and aggressive model of surgically induced osteoarthritis (Figure 5A) (28-30). 2 days after surgery, no treatment, free IGF-1, or Gen 6 45% PEG – IGF-1 was injected into the affected knee to a final joint concentration of 6 μM IGF-1 (~10 μM dendrimer). After 4 weeks, the animals were sacrificed and joint tissues were harvested. Histological processing and analysis was performed according to the guidelines for rat surgical models by the Osteoarthritis Research Society International (OARSI) histopathology initiative (33, 34) . Six 250 µm frontal step sections were taken from the central ~1.5 mm of the joint and stained with Toluidine Blue / Fast Green (Figure 5B). Aggrecan in healthy cartilage is stained blue and collagens in bone or degenerated cartilage are stained green. The section with the most severe medial tibia cartilage lesion was identified by a blinded investigator and used for all further analysis. Figure 5C shows representative sections of the medial joint for each of the 4 treatment conditions. Sections were scored quantitatively for degeneration in a blinded manner by histomorphometry techniques based on OARSI histopathology initiative guidelines for this model.

Figure 5. Dendrimer-IGF-1 conjugate reduces cartilage degeneration at 4 weeks after surgical traumatic joint injury.

(A) Schematic of a rat knee frontal section illustrating the ACLT+MMx surgery. Dotted lines outline the primary zone of lesion formation. (B) Schematic of surgery timeline and tissue processing procedures. I.A.: intra-articular (C) Representative toluidine blue / fast green stained frontal sections of the medial femur and tibia. Area of degeneration outlined in red. Total and significant widths of degeneration are outlined in black and yellow, respectively. Matrix loss shown as black arrowheads. MF: medial femur, MT: medial tibia, MM: medial meniscus, AC: articular cartilage, L: Lesion. Scale: 500 µm. (D) Quantified area of degenerated cartilage tissue of the medial tibia for each rat, as a % of total cartilage area in the section. Degenerated tissue was defined as >50% cell death and loss of toluidine blue staining. (E) Width of cartilage degeneration at the joint surface (0% depth) (F) Width of cartilage degeneration at 50% cartilage depth. (G) Representative H&E stained frontal sections of rat joints across different treatment, displaying regions of the lateral synovium characteristic of the given score (in parenthesis). Cyan arrow: increased number of synovial lining cells, black arrow: subsynovial proliferation. (H) Synovial inflammation scores (0-4) for each treatment. All data are means + 95% CIs, N = 7-9 rats as shown, statistics by one-way ANOVA and Tukey HSD post-test for (D) and (E), by Kruskal-Wallis test and Dunn post-test for (F) and (H)

Rats treated with Gen 6 45% PEG – IGF-1 after surgery showed mean degeneration of 8.4% of medial tibial cartilage area (Figure 5D). This was significantly less (p = 0.017) than the untreated group, with a mean area degeneration of 23.7%. Free IGF-1 reduced area degeneration to 19.7%, but this was not significantly different from the untreated control. Similar trends were seen for widths of degeneration (Figure 5E). At the cartilage surface (0% tissue depth), the mean width of degenerated tissue among rats treated with Gen 6 45% PEG – IGF-1 was 661 µm, compared to 1390 µm for IGF-1 alone and 1640 µm for no treatment. Surface degeneration following Gen 6 45% PEG – IGF-1 treatment was statistically less than both groups (p = 0.0023 vs untreated, p = 0.020 vs free IGF-1) and statistically equivalent (p = 0.57) to the sham operation, with a mean surface degeneration of only 343 µm. Similar trends were seen for later stage metrics of joint degeneration, such as degeneration width at 50% tissue depth (Figure 5F), but differences between treatments were not significant. Data for these late stage disease metrics appear to be bimodal, with a number of joints not exhibiting any deep degeneration or matrix loss across all treatment groups, suggesting that the disease had not yet progressed to those stages of damage in many animals at 4 weeks after surgery.

Synovial inflammation in the lateral joint capsule (Figure 5G) was assessed by a blinded, board certified pathologist and scored on a semi-quantitative 0-4 scale based on OARSI histopathology initiative guidelines for this model, taking into account number of synovial lining cell layers, subsynovial tissue proliferation, and inflammatory cell infiltrates (Figure 5H). Synovial inflammation was relatively low overall at 4 weeks post-surgery, though untreated and free IGF-1 treated rat joints had a significantly higher inflammation score than the sham surgery (p = 0.029 and 0.048, respectively). Dendrimer-IGF-1 treated joints had a mean score nearly 1 point lower than untreated joints, 0.8 points lower than free IGF-1, and 0.3 higher than the sham operation.

Dendrimer – IGF-1 reduces osteophyte burden in a surgical rat model

Following animal sacrifice, excised joints were imaged by micro-computed tomography (µCT). Osteophytes were identified and traced serially in 2D frontal sections in a blinded manner following the methods of Batiste et al. (35, 36), based on protrusion from the normal bone contour and reduced bone mineral density. Figure 6A shows representative 2D µCT image sections with osteophytes identified by arrows, as well as 3D reconstructions of the images with the full volume of the osteophyte shaded red. Total osteophyte volume was quantified for each rat (Figure 6B). Rats that received surgery and no treatment showed high variability in osteophyte formation, with severe osteophytes in some rats and minor osteophytes in others. A single injection of free IGF-1 following surgery substantially reduced osteophyte burden by nearly 50% (1.10 to 0.60 mm³) but this effect was not statistically significant (p = 0.14). A single injection of Gen 6 45% dendrimer IGF-1 was even more effective, resulting in a mean total osteophyte burden of 0.23 mm³, which constituted a statistically significant reduction (p = 0.004) from untreated rats and was nearly equivalent to osteophyte burden of sham operated rats (0.17 mm³, p = 0.99).

Figure 6. Dendrimer-IGF-1 conjugate reduces osteophyte burden in surgically injured rats to sham levels at 4 weeks post surgery.

(A) Representative 2D (top) and 3D (bottom) µCT images showing osteophytes in red arrows or red shading. ROIs were serially drawn around osteophytes in sequential frontal image stacks and reconstructed into 3D to generate bottom images and measure osteophyte volume (B) Total osteophyte volume in each joint across the four treatment conditions. Data are means + 95% CI, N = 7-9 rats as shown, statistics by one way ANOVA and Tukey HSD post-test.

Discussion

There is tremendous unmet medical need for a disease-modifying osteoarthritis drug. Anabolic growth factors, such as BMP-7, FGF-18, and IGF-1, have shown potential for disease modification by decreasing chondrocyte loss and increasing matrix production in preclinical studies(12, 24, 37-39). Anabolic disease-modifying strategies are particularly interesting in light of a recent finding by Heinemeier et al. signifying that cartilage renewal in adults is driven by aggrecan proteoglycan synthesis, with the underlying collagen matrix exhibiting no renewal in both healthy and osteoarthritic joints (40). Thus, the use of growth factors to provide enhanced production of aggrecan and protection of the chondrocytes that produce it seems a viable strategy for disease modification. Clinically, intra-articular FGF-18 increased cartilage thickness relative to placebo in a phase II OA trial (NCT01033994). However, the primary endpoint of the trial was not met (41). As free drugs, these growth factors and other biologics are hindered in efficacy by their short intra-articular half-lives and limited penetration of cartilage (9, 39, 42).

In vivo imaging and penetration studies in Figures 3 and 4 confirmed that the intra-articular pharmacokinetics of free IGF-1 are indeed quite poor. With a joint half-life of just over 10 hours, even a relatively high 6 μM joint concentration will begin losing effect within 3 days. Moreover, IGF-1 could not penetrate more than ~20 μm deep into human thickness tissue (~1000 um), thus exposing only a small number of the chondrocytes that must be treated for therapeutic gain.

Cartilage penetration is a crucial yet often neglected translational consideration in cartilage drug delivery. Any disease-modifying drug targeting chondrocytes must penetrate 1000-2000 µm of cartilage in humans to access all resident chondrocytes (43). However, many studies in the cartilage delivery field focus solely on mouse or rat cartilage, which is about 10 times thinner, between 50-200 μm depending on location in the joint (44-46). As an avascular extracellular matrix, diffusion is the primary mode of transport available through cartilage, the rate of which can be enhanced by a factor of two by dynamic compression, such as that induced by joint motion (32). A given material will take a couple orders of magnitude greater time to diffuse through human tissue compared to rodent tissue based on tissue thickness alone. Thus, demonstrating penetration through thicker tissues, such as bovine cartilage, is absolutely necessary for translation of cartilage drug delivery technology intended to target chondrocytes (47). Some evidence of this discrepancy between rodents and larger mammals in our study can be observed by comparing data on the penetration of IGF-1 through rat and bovine cartilage. Free IGF-1 was observed within cartilage 2 days after injection into rat knees (Figure S10) but was fully cleared from the tissue by 6 days (Figure 3F). In 6 days, free IGF-1 could only penetrate ~20 µm through bovine cartilage (Figure 4B), suggesting that if free IGF-1 were injected into a larger mammal like a cow or human, it would be cleared well before it could access more than ~1-2% of its target tissue.

Here, we presented a modified dendrimer nanocarrier capable of extending IGF-1 intra-articular half-life tenfold and enabling full penetration of human thickness cartilage, thereby maintaining therapeutic quantities of the growth factor in cartilage for 30 days. We hypothesized that optimal drug delivery would improve IGF-1 efficacy and validated this hypothesis in a rat model of OA with a high hurdle for detecting therapeutic cartilage protection (48). A single injection of free IGF-1 was unable to show statistically meaningful improvement in osteoarthritic rat knee joints. The Gen 6 45% PEG dendrimer – IGF-1 conjugate provided significant reduction in degenerated cartilage area, degenerated surface cartilage width, and total osteophyte volume relative to untreated rats, by factors of 3, 2.5, and 5 respectively. Moreover, Gen 6 45% PEG – IGF-1 treated rats were statistically equivalent to sham operated rats by these metrics.

The partially PEGylated dendrimer-drug conjugate possesses several translational advantages over other cartilage drug delivery systems. PEG and PAMAM dendrimers are already manufactured at kilogram scales. PEG is a component in a wide variety of marketed pharmaceuticals. PAMAM and other cationic dendrimers have been used safely in clinical trials (NCT01577537, NCT03500627, EUDRACT 2016-000877-19). The final PEGylated dendrimer-drug conjugate can be purified and characterized by standard chemical techniques, such as liquid chromatography, NMR, and MALDI-TOF mass spectrometry (Figures S2, S3, Tables S1, S2). This system is particularly suited for delivery in charged matrices with small mesh size, such as cartilage, due to both its adaptable charge and small size compared to other nanocarriers.

We have shown that the PEGylated dendrimer can incorporate biologic cargo without loss in bioactivity (Figure 1), making it ideal for delivery of biologics in the intra-articular space. The nondegradable bioconjugation scheme used is particularly advantageous for IGF-1 delivery, as it is designed to sterically hinder the N terminus of IGF-1, where the binding site for cartilage IGF-1 binding proteins is located. These IGF-1 binding proteins sequester IGF-1 from its target cellular receptors and are upregulated with age and OA; they are thought to be responsible for a decrease in responsiveness to IGF-1 observed in aged or diseased rats and primates, including humans (49-52). The bioconjugation chemistry used is flexible and ostensibly could be applied to nearly any molecule possessing a suitable synthetic handle. However, in order to incorporate a drug with an intracellular target, including nucleic acids and most small molecule therapeutics, a biodegradable moiety should be introduced into the dendrimer-drug linker.

Tunable surface charge is another key feature of the partially PEGylated dendrimer-drug conjugate system. Prior work in our groups has shown that positively charged nanocarriers bind and penetrate cartilage efficiently via reversible electrostatic interactions with anionic cartilage matrix proteoglycans (see Figure 1A) (11, 12, 15). Yet, the effects of degree of charge on these delivery properties have not been thoroughly explored. We systematically varied dendrimer surface charge by adjusting fractional end group PEGylation (0-60%) and observed dramatic effects on cartilage binding and cytotoxicity (Figure 2D). Gen 6 45% PEG, which has 3 times as many free surface amines as Gen 4 35% PEG, exhibited greater total uptake into cartilage but slower tissue penetration (Figure 4), as expected based on fundamental combined diffusion & binding transport kinetics. The more charged Gen 6 45% PEG formulation possessed a longer intra-articular half-life than Gen 4 35% PEG (Figure 3).

Importantly, the partially PEGylated dendrimer system enables surface charge to be chemically tuned, characterized, and tested for biological performance by the methodology shown in Figure 2A. This feature enables the understanding of structure-property relationships in cartilage drug delivery. This platform approach provides the opportunity to optimize these systems for a range of intra-articular delivery goals based on desired therapeutic release profiles.

The highest performing PEGylated dendrimer-IGF-1 conjugate that we identified in this study, Gen 6 45% PEG – IGF-1, showed no signs of toxicity at the cellular, tissue, or organ level (Figure 2D-F, S8, Table S3). Unmodified PAMAM dendrimers have been shown to be toxic in a dose- and generation-dependent manner, and this was consistent with our results (53) (Figure 2D, S5, 0% PEG). However, there are numerous reports of PEGylation at terminal amines mitigating or even nullifying this toxicity (18, 54, 55). Our results were consistent with these findings. The negatively charged proteoglycans present in high quantities in cartilage and joint synovial fluid may provide some additional protection from cationic toxicity in this application. Local delivery by intra-articular injection enables the use of relatively small quantities of dendrimer and its associated drug, which mitigates concerns of systemic toxicity.

It is important to note the limitations of this study as well as additional questions that need to be investigated prior to clinical translation of this work. Although systemic biodistribution of these PEGylated dendrimers was not measured in this study, systemic bioelimination of 60% end group ³H acetylated Gen 5 PAMAM dendrimers has been investigated by Nigavekar et al. (56). Within 7 days, 30% of total injected dose (ID) was excreted via urine and 3% was excreted via feces. The PEGylated dendrimer in this study have a greater hydrodynamic radius, thus excretion can be expected to shift from urine to feces. Nigavekar et al. found that most organ uptake was localized to the liver, kidneys, and lungs at around 1-2 % ID/g tissue at 7 days; these numbers decreased to 0.2-1.1 % ID/g by 12 weeks post-injection. We evaluated histological sections of these organs at 8 weeks post-injection and found no signs of histotoxicity (Figure S8).

While the rat ACLT+MMx is a well characterized, rigorous model of osteoarthritis (28-30, 48), preclinical investigation in larger animals is necessary for regulatory approval. This is especially important given the aforementioned need to demonstrate penetration of thick cartilage in vivo.

In this study, animals were sacrificed for histological evaluation of cartilage at 4 weeks, which coincided with both the duration of therapy provided by a single injection of Gen 6 45% PEG –IGF-1 and the reported onset of significant differences in tissue degeneration and osteophyte formation between operated and sham joints (29, 48). At this timepoint, we did not observe matrix loss or subchondral bone pathology in the majority of histological samples and so did not measure these features. Future animal studies should incorporate multiple injections over 8-12 weeks to observe the effect of this treatment on these late stage endpoints.

In summary, we have identified a cationic nanoformulation capable of enhancing drug therapeutic lifetime to 30 days and cartilage penetration to at least 1 mm within articular joints. The dendrimer-IGF-1 formulation enhanced the efficacy of IGF-1 in protecting both cartilage and bone in a rat surgical model of osteoarthritis, reducing the total area and width of medial tibial cartilage degeneration as well as total volume of osteophytes in the joint. The optimally PEGylated dendrimer-IGF-1 conjugates did not show any signs of toxicity on the cellular, tissue, organ, or organism levels. Importantly, the pharmacokinetics of the nanoformulation are acceptable for repeat intra-articular injection (~ monthly) and unlike the majority of nanocarriers reported for osteoarthritis, the formulation is capable of penetrating human thickness cartilage. The chemistry used in dendrimer-drug conjugation is amenable to multiple classes of drugs, including small proteins, antibodies, small molecules, and nucleic acids. These results suggest the possibility of improving the efficacy of osteoarthritis drugs of the past and future by utilizing a partially PEGylated dendrimer-drug conjugate. This cartilage penetrating nanocarrier could rejuvenate the field of osteoarthritis drug development and accelerate the discovery of the first disease-modifying osteoarthritis drug.

Materials and Methods

Study Design

Dendrimer-IGF-1 conjugates were evaluated for their pharmacokinetics in knee joints and efficacy in treating knee post-traumatic osteoarthritis relative to free IGF-1. Surgical study outcomes used to evaluate therapeutic efficacy were prospectively designed to include both cartilage and bone pathology using histomorphometry and µCT imaging, respectively. We chose to evaluate joints at 4 weeks post-surgery in order to evaluate both inflammatory response and early tissue pathology, according to characterization of osteoarthritis development in this model (29, 30). Moreover, our data showed that a single injection of dendrimer-IGF-1 remains active in the joint for around 4 weeks. Prospective power analysis was performed using G*Power Analysis and statistical analysis was performed using appropriate tests and multiple hypothesis testing corrections as indicated in the figures. Effect sizes were estimated based on prior experiments in our group or published literature. Probability of type I and type II error were set at 0.05 and 0.20, respectively. OA severity in this model is strongly affected by surgical skill, and so subject order was blocked, not randomized, to account for this known variable. All histological and µCT measurement and scoring was conducted by a blinded investigator. Identifying animal numbers were converted to an unrelated system of letters by an individual uninvolved in scoring and were not revealed until all data analysis was complete. Six total animals were removed from histomorphometric analysis due to widespread damage across the medial tibia, rendering quantitative measurement of cartilage impossible. A pathologist confirmed that this damage was mechanical in nature, likely due to severe joint instability generated by unintentional transection of multiple joint ligaments in the surgery. Joints were excluded while the investigators were blinded and were distributed across the 3 non-sham treatment groups.

Dendrimer-IGF-1 Bioconjugation

PAMAM dendrimers, G4 and G6, were obtained from Dendritech through Sigma. 350 nmol PAMAM was reacted with N-Hydroxysuccinimide (NHS) methyl-PEG₈ (mPEG₈-SCM, Creative PEGWorks) and NHS-maleimide-PEG₁₂ (SMPEG₁₂, Thermo-Fisher) in 0.1 M NaHCO₃ buffer, pH 8.0. In a separate pot, Alexa Fluor 647 (AF647, Life Technologies) and N-Succinimidyl S-Acetylthioacetate (SATA, Thermo-Fisher) in anhydrous DMSO were added to 1 mg/mL IGF-1 (Biovision), each in a 2.5:1 molar ratio to produce IGF-1-SATA labeled with AF647. Both reactions were run for 2 hours at 25°Cw ith rotation, protected from light. Maleimide functionalized PEGylated dendrimers were purified by ultracentrifugation (Amicon Ultra, Millipore) using 0.1 M phosphate buffer, pH 6.8 and IGF-1-SATA was purified on a dextran desalting size exclusion column (5 kDa, Thermo-Fisher) with 0.1 M phosphate buffer as the eluent. The IGF-1-SATA was deacetylated to form IGF-SH by addition of 10 v/v% 0.5 M hydroxylamine (Thermo-Fisher) in 0.1 M phosphate buffer w/ 0.01 M EDTA, pH 8.0 for 50 minutes, then purified with a dextran size exclusion column using 0.1 M phosphate buffer w/ 0.01 M EDTA, pH 6.8 as the eluent.

50 nmol of maleimide functionalized PEGylated dendrimer was reacted with ~ 65 nmol IGF-1-SH for 48 hours at 4°C with stirring. The reaction was then quenched by adding 2 μmol cysteine for 2 hours. The mixture was purified by cationic exchange chromatography using a salt gradient at pH 6.8. Approximately 60% of dendrimers were conjugated to IGF-1. IGF-1 yield based on starting quantity was about 25%.

Bovine Cartilage Explant Harvest and Culture

Young (1-2 weeks old) bovine knee joints were obtained from Research 87 (Hopkinton, MA) and cartilage was harvested from the trochlear groove and cultured by the methods in (24). Treatment conditions were blocked across explant location across the joint surface.

Cartilage Explant Penetration

750 uL of 10 μM near-IR fluorescent (Alexa Fluor 647) PEGylated dendrimer formulation (Gen 4 35% - IGF-1, Gen 6 45% - IGF-1, or free IGF-1) in DMEM (w/o phenol red) + 10% v/v FBS + 1% v/v Pen-Strep was added to 6×1 mm cartilage explants. The explants were incubated for 48 or 144 hours at 37°C and 5% CO₂ under gentle agitation. After incubation, cartilage explants were washed in PBS and sectioned to 80 μm using a vibrating microtome (Leica Biosystems). Sections were mounted on glass slides and immediately observed under a confocal fluorescent microscope (Nikon A1R Scanning Confocal Microscope). Quantitative analysis was performed on maximum intensity projections of z-stack images taken of the 80 μm sections.

Intra-Articular Pharmacokinetics

12 week old male Sprague-Dawley rats were anesthetized under isoflurane, shaved on both legs, and disinfected with 3 scrubbing routines of povidone iodine followed by 70 v/v% ethanol. 40 μL of Gen 4 35%-IGF-1, Gen 6 45% IGF-1, or free IGF-1, all matched in IGF-1-Alexa 647 concentration (7.3 μM AF647, ~ 20 μM IGF-1), in sterile isotonic saline were injected into both legs of the rat intra-articularly. An In Vivo Imaging System (IVIS Spectrum, Perkin Elmer) and Living Image software (Caliper) were used to serially acquire and quantify fluorescence within each joint over a period of 28 days. Radiant efficiency data (units shown in Equation 1, below) within a fixed anatomical R.O.I. over time for each formulation type were fit to a single phase exponential decay with a common plateau based on untreated animal background fluorescence.

$$\mathrm{Radiant}\mathrm{Efficiency}\mathrm{units}:\left(\frac{{}^p∕_{\mathit{sec}\ast {\mathit{cm}}^2\ast \mathit{sr}}}{{}^{\mu W}∕_{{\mathit{cm}}^2}}\right)$$ (1)

In a separate experiment under the same injection protocol, the distribution of fluorescently labeled drug or dendrimer-drug conjugate was observed in rat knees 2 and 6 days after injection. Joints were excised, split into tibia and femur, stained with a fluorescent antibody against aggrecan (Thermo-Fisher), and imaged intact at the medial femoral condyle and medial tibial plateau in an Olympus FV1000 multiphoton confocal microscope throughout the depth of the cartilage tissue (120-160 μm) using an 840 nm Ti:sapphire laser and a 25x water objective with an NA of 1.25.

Rat Surgical Osteoarthritis Model: ACLT+MMx

All animal work was performed in accordance with protocols approved by the Committee on Animal Care (IUCAC) at MIT as well as the Animal Care and Use Review Office (ACURO) at the U.S. Army Medical Research and Materiel Command. Skeletally mature male Sprague-Dawley rats of 12 weeks of age (350-400 g, Taconic) were anesthetized by isoflurane, injected with 1 mg/kg subcutaneous Meloxicam and Buprenorphine Sustained Release (Bup-SR), and given eye ointment. The right hind limb was shaven and disinfected with povidone iodine scrub followed by 70 v/v% ethanol in water. The rat was transferred to an operating table, rested on a heated recirculating water pad, and draped in a sterile manner. A shallow medial parapatellar incision was made with a #15 scalpel to expose the joint capsule, followed by a second incision to open the joint capsule. The incision was extended with iris scissors until the patella could be subluxated laterally, exposing the joint. Bleeding was mitigated by lavage with sterile saline and dabbing with sterile gauze. The ACL was exposed and identified by flexing the knee acutely and utilizing surgical loupes. At this point, the sham operation was complete. For animals subjected to transection & meniscectomy, the ACL was transected with vannas microscissors and transection was confirmed by the anterior drawer test. The medial meniscus was partially transected by inserting a #11 blade beneath the midpoint of the femoral condyle and pressing down gently on the tibia. Following the completion of the procedure, the joints were thoroughly washed with sterile saline and the patella was relocated. The joint capsule and skin were each closed separately with 5-0 polyglecaprone resorbable sutures. The skin sutures were reinforced with stainless steel wound clips and tissue glue. Following surgery, rats were given 20 mL/kg warm subcutaneous saline, housed singly, allowed unrestricted movement, and put on an analgesic regimen of q24 1 mg/kg subcutaneous Meloxicam and q48 Bup-SR for 3 days.

Rats were divided into 4 groups: Transection and no treatment (untreated), Transection and free IGF-1 treatment (free IGF-1), Transection and Gen 6 45% PEG – IGF-1 treatment (Gen 6 45% - IGF-1), and sham surgery (Sham). Treatments were given by intra-articular injection into the affected joint 48 hours after surgery.

Histopathology

Processing of tissues for histology is presented in the SI and followed the methods of Schmitz et al.(33). The anterior and posterior tissues were embedded in the same cassette in paraffin, and sectioned into a number of 5 μm frontal sections at 3 250 μm steps within the joint. Sections were stained with Toluidine Blue / Fast Green and the section containing most severe medial tibial lesion was identified by a blinded investigator for scoring and IHC. Gross pathological scoring followed the published guidelines of the Osteoarthritis Society International (OARSI) Histopathology Initiative (Gerwin et al., 2010) (34) and was performed quantitatively using ImageJ (NIH) overlays on scanned digital slides (Leica) by a blinded investigator (BCG). Semi-quantitative synovial inflammation grading also followed OARSI guidelines (34) and was performed by a blinded, board-certified pathologist (RFP).

Micro-Computed Tomography

After fixation and before decalcification, excised rat joints were attached to the bore of a microCT scanner (GE eXplore CT 120) and scanned at .03° angles for a total of 1200 scans over a time period of 1.5 hours. Scans used an 80 kV x-ray potential, 32 mA current, and a 100 ms integration time. Image data sets were reconstructed into regions of interest encompassing single joints using Microview software (Parallax Innovations).

3D positioning data were used to quantify total volume of osteophytes on each joint. 3D DICOM analysis software (Osirix MD) was used by a blinded investigator (BCG) to re-section the images into a uniform frontal plane, perpendicular to an axis tangential to the femoral condyles and tibial plateau in the sagittal plane and an axis bisecting the femoral condyles and tibial plateau in the transverse plane. The blinded investigator then manually outlined osteophytes with regions of interest (ROIs) in each contiguous frontal section. Osteophytes were defined as bone exhibiting both reduced bone mineral density and protrusion from the normal bone contour. The sections were then reconstructed to 3D, and total osteophyte ROI volume was calculated (35, 36).

Statistics

For all experimentation shown, error bars indicates 95% confidence intervals. Statistical analyses were performed with Graphpad Prism and SAS JMP. Two sided p values less than 0.05 were considered statistically significant. Parametric data were analyzed by ANOVA methods followed by Tukey HSD post-hoc tests. Nonparametric data were analyzed by a Kruskal-Wallis test followed by Dunn post-hoc tests. Exponential decay constants from Fig. 3 were tested for statistical difference using the extra sum of squares F test, with a null hypothesis that data from all three treatments were adequately fit by a simple model with a common decay constant. Multiple hypothesis testing corrections are indicated in figure legends, if applied.

Supplementary Material

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Fig. S1. Representative cell cycle flow cytometry scatter plots for NIH 3T3 cells after 24 hour treatment with IGF-1 formulations

Fig. S2. ¹H-NMR spectra of PEGylated dendrimer panel (without IGF-1)

Fig. S3. MALDI-TOF mass spectra for partially PEGylated dendrimer screen

Fig. S4. Complete uptake data for Generation 4 and Generation 6 partially PEGylated dendrimers into bovine cartilage explants.

Fig. S5. Complete CHON-001 cellular viability data for (top) Generation 4 and (bottom) Generation 6 partially PEGylated dendrimers at a range of doses of dendrimers.

Fig. S6. Desorption of PEGylated dendrimers in solutions of different ionic strength

Fig. S7. Quantification of ex vivo bovine cartilage tissue viability staining in Figure 2

Fig. S8. Histology (H&E) of representative sections from likely contact organs for ~5-10 nm materials

Fig. S9. Histology (H&E) of rats treated with dendrimers having sub-optimal % end group PEGylation

Fig. S10. 3D reconstruction of multiphoton microscopy images of full thickness cartilage from intact rat femurs harvested 2 days post-injection of free IGF-1

Fig. S11. Penetration of cartilage explants in DMEM (no protein in media)

Table S1. Characterization of % PEGylation by NMR integral ratios

Table S2. Characterization of % PEGylation by mass addition according to MALDI-TOF data

Table S3. Serum toxicology biomarker levels at 2 and 7 days post-intraarticular injection of Gen 6 45% PEG – IGF-1