Integration of Phase-Change Materials with Electrospun Fibers for Promoting Neurite Outgrowth under Controlled Release

Abstract

We report a temperature-regulated system for the controlled release of nerve growth factor (NGF) to promote neurite outgrowth. The system is based upon microparticles fabricated using coaxial electrospray, with the outer solution containing a phase-change material (PCM) and the inner solution encompassing payload(s). When the temperature is kept below the melting point of the PCM, there is no release due to the extremely slow diffusion through a solid matrix. Upon increasing the temperature to slightly pass the melting point, the encapsulated payload(s) can be readily released from the melted PCM. By leveraging the reversibility of the phase transition, the payload(s) can be released in a pulsatile mode through on/off heating cycles. The controlled release system is evaluated for potential use in neural tissue engineering by sandwiching the microparticles, co-loaded with NGF and a near-infrared dye, between two layers of electrospun fibers to form a tri-layer construct. Upon photothermal heating with a near-infrared laser, the NGF is released with well-preserved bioactivity to promote neurite outgrowth. By choosing different combinations of PCM, biological effector, and scaffolding material, this controlled release system can be applied to a wide variety of biomedical applications.

Keywords: coaxial electrospray; controlled release; electrospun fibers; neural tissue engineering; phase-change material.

Figure 1.

(A) Schematic illustration of the coaxial electrospray setup used for the fabrication of PCM microparticles containing a payload in the core region. (B, C) Optical and fluorescence micrographs, respectively, of the microparticles loaded with Rhodamine B. (D, E) Fluorescence micrograph and SEM image, respectively, of the microparticles loaded with FITC-BSA.

Figure 2.

Release of various types of payloads from the PCM microparticles. (A) Cumulative release of FITC-BSA from the particles upon continuously heating at 37 °C and 40 °C, respectively (n = 3). (B) Amounts of HRP released from the particles after heating at 37 °C and 40 °C for 3 min, respectively, and repeated for five rounds (n = 3). (C) UV/vis absorption spectra of the blue-colored product derived from the reaction between TMB and the HRP released from the particles by heating at 40 °C for 3 min. The spectra were recorded for 10 min at an interval of 30 s. (D) Typical plot of the time-dependent absorbance changes of the blue-colored product at 650 nm derived from the reaction between TMB and the native or thermally released HRP, respectively. At the same concentration of HRP, the slower initial reaction rate constant, which is the slope of the initial linear region, suggests a lower enzymatic activity.

Figure 3.

Photothermal effect and the release of payloads from the PCM microparticles. Indocyanine green (ICG) was co-loaded into the particles as a NIR absorber. (A) Infrared images showing the rise of temperature to slightly pass the melting point of the PCM upon irradiation with the diode laser at different power densities for different periods of time. Cumulative release of (B) FITC-BSA and (C) HRP from the particles upon laser irradiation at a power density of 1.0 W/cm², respectively (n = 3). (D) Typical plot of the time-dependent absorbance changes of the blue-colored product at 650 nm derived from the reaction between TMB and the native or photothermally released HRP, respectively.

Figure 4.

The cumulative release profiles of NGF from the tri-layer construct upon (A) direct heating at 37 °C and 40 °C, respectively, for 3 min and repeated six rounds (n = 3) and (B) photothermal heating upon laser irradiation for 6 s at a power density of 1.0 W/cm² and repeated six rounds (n = 3).

Figure 5.

Fluorescence images of the PC12 cells after incubation for 7 days under the effect of different constructs upon laser irradiation: (A) bi-layer construct with one layer consisting of random fibers and the other layer consisting of random fibers, (B) tri-layer construct sandwiched with PCM microparticles containing ICG only (PCM-ICG), (C) bi-layer construct with free NGF supplemented in the culture medium, (D) tri-layer construct (PCM-ICG) with free NGF supplemented in the culture medium, and (E) tri-layer construct sandwiched with PCM microparticles containing both ICG and NGF (PCM-ICG/NGF). (F) Fluorescence image of the PC12 cells after incubation for 7 days under the effect of tri-layer construct sandwiched with PCM microparticles containing both ICG and NGF (PCM-ICG/NGF) in the absence of laser irradiation. The plasma membrane was stained in red while the cell body was stained in green using a Neurite Outgrowth Kit. Scale bar = 50 μm. (G) The average and the longest lengths of neurites extending from the PC12 cells, and (H) the percent of cells bearing neurites. Measurements were taken from 3 sets of 150 cells for each group. The three sets were from three representative experiments. ^#P < 0.05 significantly higher than the group of bi-layer construct; by Student’s t test. **P<0.01 compared with the group with no laser irradiation, by Student’s t test.

Figure 6.

(A) Schematic showing the neurite outgrowth from spheroids of PC12 cells incubated on the tri-layer construct under the effect of NGF released from the sandwiched PCM particles upon photothermal heating with the diode laser. Fluorescence micrographs of the typical neurite fields extending from the spheroids when directly cultured on the tri-layer construct in the (B) absence and (C) presence of laser irradiation, respectively. The neurites were stained with Tuj1 marker (green). The arrows indicate the alignment directions of the fibers.

Figure 7.

The average and the longest lengths of neurites extending from the spheroids of PC12 cells when directly cultured on the tri-layer construct with/without laser irradiation. **P<0.01 when compared with the group without laser irradiation, by Student’s t test (n = 3).