The controlled release of active substances—bone morphogenetic protein 2 (BMP-2) and 17β-estradiol—is one of the main aspects to be taken into account to successfully regenerate a tissue defect. In this study, BMP-2- and 17β-estradiol-loaded microspheres were combined in a sandwich-like system formed by a hydrogel core composed of chitosan (CHT) collagen, 2-hidroxipropil γ-ciclodextrin (HP-γ-CD), nanoparticles of hydroxyapatite (nano-HAP), and an electrospun mesh shell prepared with two external electrospinning films for the regeneration of a critical bone defect in osteoporotic rats. Microspheres were made with poly-lactide-co-glycolide (PLGA) to encapsulate BMP-2, whereas the different formulations of 17β-estradiol were prepared with poly-lactic acid (PLA) and PLGA. The in vitro and in vivo BMP-2 delivered from the system fitted a biphasic profile. Although the in vivo burst effect was higher than in vitro the second phases (lasted up to 6 weeks) were parallel, the release rate ranged between 55 and 70 ng/day. The in vitro release kinetics of the 17β-estradiol dissolved in the polymeric matrix of the microspheres depended on the partition coefficient. The 17β-estradiol was slowly released from the core system using an aqueous release medium (D_eff = 5.58·10⁻¹⁶ ± 9.81·10⁻¹⁷m²s⁻¹) and very fast in MeOH-water (50:50). The hydrogel core system was injectable, and approximately 83% of the loaded dose is uniformly discharged through a 20G needle. The system placed in the defect was easily adapted to the defect shape and after 12 weeks approximately 50% of the defect was refilled by new tissue. None differences were observed between the osteoporotic and non-osteoporotic groups. Despite the role of 17β-estradiol on the bone remodeling process, the obtained results in this study suggest that the observed regeneration was only due to the controlled rate released of BMP-2 from the PLGA microspheres.

Lipid-polymer hybrid nanoparticles are promising platforms in the field of targeted drug delivery, integrating the positive features of polymeric and lipid nanocarriers. However, the use of bulk procedures in lipid-polymer hybrid nanoparticles formulation is hindering their large-scale manufacturing. Therefore, the aim of this study is to explore the suitability of alternative formulation methods, such as microfluidics, to obtain surface-tunable nanoparticles displaying suitable characteristics. Formulations were prepared by single-step nanoprecipitation or using a micromixer chip. The nanocarriers were then surface-modified with an aptamer and an antibody, two common nanoparticle vectorization strategies, developing an optimized functionalization protocol. Both naked and surface-modified nanoparticles were characterized in terms of size, polydispersity, zeta potential and morphology. Moreover, the aptamer/antibody association efficiency was also determined. Nano-sized monodisperse nanoparticles, exhibiting a spherical core-shell structure, were obtained through both procedures. Furthermore, all the nanocarriers were successfully functionalized, showing association efficiency values above 70%. Interestingly, microfluidic-based nanoparticles displayed a smaller size and a more positive zeta potential than those prepared by single-step nanoprecipitation. Outcomes suggest both techniques led to lipid-polymer hybrid nanoparticles displaying a similar functionalization efficiency. Conversely, the microfluidic approach provided an improved control over critical parameters, as particle size or charge, constituting an interesting alternative to traditional formulation procedures.

Osteoporosis (OP) is characterized by a loss in bone mass and mineral density. The stimulation of the canonical Wnt/β-catenin pathway has been reported to promote bone formation, this pathway is controlled by several regulators as secreted frizzled-related protein-1 (Sfrp-1), antagonist of the pathway. Thus, Sfrp-1 silencing therapies could be suitable for enhancing bone growth. However, the systemic stimulation of Wnt/β-catenin has been correlated with side effects. This work hypothesizes the administration of lipid-polymer NPs (LPNPs) functionalized with a MSC specific aptamer (Apt) and carrying a SFRP1 silencing GapmeR, could favor bone formation in OP with minimal undesired effects. Suitable SFRP1 GapmeR-loaded Apt-LPNPs (Apt-LPNPs-SFRP1) were administered in osteoporotic mice and their biodistribution, toxicity and bone induction capacity were evaluated. The aptamer functionalization of the NPs modified their biodistribution profile showing a four-fold increase in the bone accumulation and a ten-fold decrease in the hepatic accumulation compared to naked LPNPs. Moreover, the histological evaluation revealed evident changes in bone structure observing a more compact trabecular bone and a cortical bone thickness increase in the Apt-LPNPs-SFRP1 treated mice with no toxic effects. Therefore, these LPNPs showed suitable properties and biodistribution profiles leading to an enhancement on the bone density of osteoporotic mice.

Gene therapy holds significant promise as a therapeutic approach for addressing a diverse range of diseases through the suppression of overexpressed proteins and the restoration of impaired cell functions. Developing a nanocarrier that can efficiently load and release genetic material into cells remains a challenge. The primary goal of this study is to develop formulations aimed to enhance the therapeutic potential of GapmeRs through technological approaches. To this end, lipid-polymeric hybrid nanoparticles (LPHNPs) with PLGA, DC-cholesterol, and DOPE-mPEG₂₀₀₀ were produced by conventional single-step nanoprecipitation (SSN) and microfluidic (MF) methods. The optimized nanoparticles by SSN have a size of 149.9 ± 18.07 nm, a polydispersity index (PdI) of 0.23 ± 0.02, and a zeta potential of (ZP) of 29.34 ± 2.44 mV, while by MF the size was 179.8 ± 6.3, a PdI of 0.24 ± 0.01, and a ZP of 32.25 ± 1.36 mV. Furthermore, LPHNPs prepared with GapmeR-protamine by both methods exhibit a high encapsulation efficiency of approximately 90%. The encapsulated GapmeR is completely released in 24 h. The LPHNP suspensions are stable for up to 6 h in 10% FBS at pH 5.4 and 7.4. By contrast, LPHNPs remain stable in suspension in 4.5% albumin at pH 7.4 for 24 h. Additionally, LPHNPs were successfully freeze-dried using trehalose in the range of 2.5-5% as cryoprotectant The LPHNPs produced by MF and SSN increase, 6 and 12 fold respectively, GapmeR cell uptake, and both of them reduce by 60–70% expression of Tob1 in 48 h.Our study demonstrates the efficacy of the developed LPHNPs as carriers for oligonucleotide delivery, offering valuable insights for their scale up production from a conventional bulk methodology to a high-throughput microfluidic technology. Graphical Abstract