Transport and equilibrium uptake of a peptide inhibitor of PACE4 into articular cartilage is dominated by electrostatic interactions

Abstract

The availability of therapeutic molecules to targets within cartilage depends on transport through the avascular matrix. We studied equilibrium partitioning and non-equilibrium transport into cartilage of Pf-pep, a 760 Da positively charged peptide inhibitor of the proprotein convertase PACE4. Competitive binding measurements revealed negligible binding of Pf-pep to sites within cartilage. Uptake of Pf-pep depended on glycosaminoglycan charge density, and was consistent with predictions of Donnan equilibrium given the known charge of Pf-pep. In separate transport experiments, the diffusivity of Pf-pep in cartilage was measured to be approximately 1 x 10(-6) cm(2)/s, close to other similarly-sized non-binding solutes. These results suggest that small positively charged therapeutics will have a higher concentration within cartilage than in the surrounding synovial fluid, a desired property for local delivery; however, such therapeutics may rapidly diffuse out of cartilage unless there is additional specific binding to intra-tissue substrates that can maintain enhanced intra-tissue concentration for local delivery.

Figure 1

(a) Schematic diagram showing incubation of cartilage explants with labeled and unlabeled Pf-pep in the bath. (b) Transport chamber consisting of two compartments, upstream bath and downstream bath. Solutes only could transport through three 9-mm diameter cartilage slices clamped between two compartments. The downstream bath was recirculated through the detector to measure the radioactivity of downstream in real-time.

Figure 2

Concentration-dependent uptake ratio of ¹²⁵I-Pf-pep in adult bovine cartilage after 48 hr at 4°C in 1×PBS buffer. Graded amount of unlabeled Pf-pep was added with fixed amount of ¹²⁵I-Pf-pep (< 1 nM). The uptake ratio of ¹²⁵I-Pf-pep did not vary significantly with the concentration of unlabeled Pf-pep over the entire range of concentration for both L1 and L2 tissue (1-way ANOVA, p = 0.825 with L1, p = 0.831 with L2). The uptake of ¹²⁵I-Pf-pep was significantly higher in L2 compared to L1 cartilage overall (2-way ANOVA, p <0.0001). Mean ± SD (n = 4 disks per condition)

Figure 3

Dependence of the uptake ratio of ¹²⁵I-Pf-pep on (a) tissue GAG density (μg GAG content/mg tissue water) and (b) tissue hydration (mg tissue water/mg dry weight). Uptake of ¹²⁵I-Pf-pep increased with increasing tissue sGAG content and with decreasing tissue hydration (linear regression, p <0.0001). Cartilage from the deeper region (L2) generally had higher GAG density (t-test, p <0.0001) and lower hydration (t-test, p = 0.00016) than L1 tissue. Each data point represents one of the 68 cartilage specimens that comprise the data-set of Fig. 2.

Figure 4

Estimate of the net charge (Z) of Pf-pep using Donnan equilibrium theory. Donnan theory predicts that the partition coefficient of ¹²⁵I-Pf-pep will follow the power-law relation with the partition coefficient of Na⁺ by the exponent Z. The partition coefficient of Na⁺ was calculated from the measured GAG density of each individual specimen (A.5) and the partition coefficient of ¹²⁵I-Pf-pep was assumed to be equal to the measured uptake ratio in Fig 3. The solid lines are the predicted theoretical curves for Pf-pep charge (Z = 1–4). The best fit value of Z was + 2.87, which corresponds well to the known charge of Pf-pep (+3). Each data point represents one of the 68 cartilage specimens that comprise the data-set of Fig. 2.

Figure 5

Effects of trypsin depletion of GAG (TRP, 0.15M NaCl) or screening of GAG electrostatic interactions (FS, 1M NaCl) on the uptake of ¹²⁵I-Pf-pep in L2 bovine calf cartilage disks. Untreated control disks in physiological ionic strength (FS, 0.15M NaCl) showed significantly higher partition coefficients compared to the two treated groups (*p<0.0001 by 1-way ANOVA post hoc Dunnett’s test, vs. control group “FS, 0.15 M”). Mean ± SEM (n = 3 disks per condition)

Figure 6

Non-equilibrium diffusion of ¹²⁵I-Pf-pep across groups of three adult bovine cartilage disks (data shown for L1 disks), plotted as the measured downstream concentration versus time, normalized to the applied upstream concentration. At t = 50 minutes, ¹²⁵I-Pf-pep was introduced to the upstream bath. The diffusivity of Pf-pep was calculated from the measured diffusive flux of ¹²⁵I-Pf-pep (i.e., the slope of the concentration vs. time data as shown). At t = 728 minutes, a 300 μl aliquot from the upstream bath (20 ml) was transferred to the downstream bath (20 ml) to calibrate the concentration. At t = 980 minutes, unbound ¹²⁵I was added to the upstream bath to estimate the contribution of unbound ¹²⁵I to the total flux and to correct for the presence of such ¹²⁵I in the calculated diffusivity of ¹²⁵I-Pf-pep. The solid line at the end of experiment shows the predicted flux which would be present if ¹²⁵I were not added.