Porous PLGA microspheres loaded with PTH1-34 peptide for long-term treatment of OA

Abstract

Background: Osteoarthritis (OA) is a chronic disease characterized by degeneration of articular cartilage, affecting over 530 million patients worldwide. Current oral medications such as non-steroidal anti-inflammatory drugs (NSAIDs) can only alleviate symptoms and are associated with numerous adverse effects. Although teriparatide (PTH_1-34) exhibits dual functions of chondroprotection and osteogenic effects, its clinical application is significantly limited by its short biological half-life (30-60 min) and accelerated degradation within the inflammatory microenvironment of joint cavities.

Methods: Porous sustained-release microspheres (M@PTH_1-34) were fabricated using FDA-approved poly (lactic-co-glycolic acid) (PLGA) as the matrix, encapsulating PTH_1-34 within their multi-channel porous structure. Uniform microsphere preparation and high-efficiency drug loading were achieved through membrane emulsification and temperature-controlled embedding techniques. To systematically evaluate the sustained-release profile and therapeutic outcomes, both in vitro and in vivo OA models were established, enabling comprehensive analysis of cartilage repair efficacy, anti-inflammatory regulation, and immunomodulatory effects.

Results: PTH_1-34 could be efficiently loaded into microspheres after self-healing and achieve consistent release over 30 days with biological activity being maintained. In OA model rats, M@PTH_1-34 significantly improved behavioral and radiological outcomes, increased cartilage smoothness and thickness, and increased the expression of chondrogenic markers. Additionally, in vitro and in vivo safety tests revealed no significant safety issues. These findings indicate that M@PTH_1-34 holds promise as a long-lasting, cost-effective, and safe therapeutic approach for OA.

Conclusion: This study successfully developed a uniform-sized PLGA-based sustained-release microsphere system (M@PTH_1-34) that enables continuous drug release for over 30 days following single intra-articular administration. M@PTH_1-34 exerts its therapeutic effects on osteoarthritis through the following two ways: (1) Promoting cartilage repair by enhancing the chondrogenic differentiation ability of bone marrow mesenchymal stem cells (BMSCs); (2) Improve the inflammatory microenvironment of joints by inhibiting the expression of inflammatory factors (such as IL-1β) and regulating the polarization state of M1/M2 macrophages.

The translation potential of this article: The system demonstrates prominent clinical translation advantages: (1) Innovative utilization of FDA-approved PLGA carrier combined with membrane emulsification technique ensures precise size control and standardized production; (2) Localized delivery strategy achieves targeted retention within articular cavity, validated by animal studies showing no systemic exposure risks; (3) Standardized preparation process demonstrates the feasibility of industrial-scale production.

Keywords: Cartilage repair; Osteoarthritis; PTH1-34; Porous PLGA microspheres; Sustained release.

Graphical abstract

Fig. 1

Characterization of PTH_1-34-loaded sustained-release microspheres (M@PTH_1-34). A. Characterization of microspheres. The representative scanning electron microscopy (SEM) images for the pre-encapsulation state and post-encapsulation state are shown on the top-left side and the top-right side, respectively. The scale bar represents 2 μm. B. A white light CLSM image of the microspheres is shown, and the red color indicates Cy5.5-labeled PTH_1-34. The scale bar represents 2 μm. C. In vitro degradation and activity test assay flowchart. D. PTH_1-34 remained in the poly (lactic-co-glycolic acid) (PLGA) microcapsules, and its release profile during in vitro immersion in culture medium for up to 40 days is shown. IVIS Spectrum images of the PLGA microcapsules; three independent repeated tests. E. The radiant efficiency of the PLGA microcapsules was quantitatively analyzed, as shown in Fig. 1D. Data are presented as the mean ± SD of at least three replicate experiments. A.U. arbitrary units. F. PTH_1-34 activity in the supernatant collected every 5 days during the release process of the microspheres was detected; three independent tests were performed. G. Luminescence of the samples after intra-articular injection, calculated with the IVIS Spectrum imaging system. Data are presented as the mean ± SD of at least three replicate experiments. A.U. arbitrary units. H. Representative images of the Alcian blue stain of bone marrow mesenchymal stem cells (BMSCs) after chondrogenic differentiation induction; the scale bar represents 50 μm. I. Quantitative analysis of acidic mucopolysaccharide expression in Fig. 1H. J-K. The RT-qPCR results of the mRNA expression levels for Col2a1, Acan. L. Schematic diagram of in vitro evaluation of the effect of M@PTH_1-34 on IL-1β-induced chondrocytes. M-P. The RT-qPCR results of the mRNA expression levels for Col2a1, Acan, MMP13, COX2. (ns p > 0.05, ∗p < 0.05, ∗∗p < 0.01 and ∗∗∗p < 0.001).

Fig. 2

Degradation of microspheres in osteoarthritis (OA) rats and gait analysis after treatment completion. A. Schematic illustration of the in vivo experimental procedure in rats: In the modified Hulth's group, the rats underwent anterior cruciate ligament (ACL) transection and medial meniscectomy. Eight weeks after surgery, different reagents were injected, and treatment was continued for 4 weeks. In the sham surgery group, only the knee joint skin was incised, followed by skin suturing and intra-articular injection of phosphate-buffered saline (PBS). B. In vivo retention of PTH_1-34-loaded sustained-release microspheres (M@PTH_1-34) as observed via arthrography; representative IVIS Spectrum images of the mice after intra-articular injection of M@PTH_1-34 over time. C. The radiant efficiency of the samples after intra-articular injection calculated by the IVIS Spectrum imaging system. Data are presented as the mean ± SD of at least three replicate experiments. A.U. arbitrary units. D. The correlation between the in vivo and in vitro release of M@PTH_1-34. E‒K. Quantitative analysis of spatiotemporal parameters (n = 4 experimental rats per group), including stride length (E), swing time (F), swing speed (G), body speed (H), print area (I), maximum contact area (J), and maximum intensity (K). Correlation analysis was performed using Spearman's rank correlation test. Statistical analysis was performed using unpaired two-tailed Student's t tests. Data are presented as the mean ± SD. ns p > 0.05, ∗p < 0.05, and ∗∗p < 0.01.

Fig. 3

Micro-computed tomography (CT) results and histological staining results after treatment in osteoarthritis (OA) rats. A&B. Representative micro-CT images of osteoarthritic knee joints subjected to various treatments and quantification of osteophyte volume. C. Representative hematoxylin and eosin (H&E) (top), safranin O-fast green (SO&FG, middle), and toluidine blue-O (TB, bottom) stained images; the scale bar represents 100 μm. D. Calculation of the cartilage area on the basis of the staining results. E‒F. OA severity in the knee joints at week 4 after injection, as evaluated with the OA Research Society International (OARSI) and Mankin scoring systems. Statistical analysis was performed using unpaired two-tailed Student's t tests. Data are presented as the mean ± SD (n = 4), ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

Fig. 4

M@PTH_1-34 modulated the levels of cartilage metabolism-related proteins in osteoarthritic joints. A. Representative images of expression of proteins related to cartilage synthesis; B. Representative images of expression of proteins related to cartilage degradation. The different pseudocolors correspond to the respective labeled proteins: green (Aggrecan), red (Col2a1), indigo (SOX9), purple (MMP13), and yellow (ADAMTS4); the scale bar represents 100 μm C. Representative images of expression of IL-1β. D. Representative images of expression of CD86 and CD163. The different pseudocolors correspond to the respective labeled proteins: green (CD163), red (IL-1β/CD86); the scale bar represents 100 μm. The phosphate-buffered saline (PBS) group was used as a reference, and protein quantification was performed using ImageJ. Statistical analysis was performed using unpaired two-tailed Student's t tests. Data are presented as the mean ± SD (n = 4), ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

Fig. 5

In vitro and in vivo safety evaluation of M@PTH_1-34. A. Representative fluorescence images of live/dead-stained bone marrow mesenchymal stem cells (BMSCs) cultured with different concentrations of M@PTH_1-34, PTH_1-34 (120 μg/ml), and empty poly (lactic-co-glycolic acid) (PLGA); green represents live cells, and red represents dead cells. Scale bar represents 100 μm. B. Statistical analysis of cell cytotoxicity. C. Representative images of hematoxylin and eosin (H&E)-stained heart, liver, spleen, lung, and kidney samples. D-H. Levels of metabolic indicators reflecting liver function (ALT and AST) and kidney function (UREA and CREA) and blood calcium levels. Statistical analysis was performed using one-way analysis of variance (ANOVA) with Tukey's post hoc test. Data are presented as the mean ± SD. ns p > 0.05.