Encapsulation-free controlled release: Electrostatic adsorption eliminates the need for protein encapsulation in PLGA nanoparticles

Abstract

Encapsulation of therapeutic molecules within polymer particles is a well-established method for achieving controlled release, yet challenges such as low loading, poor encapsulation efficiency, and loss of protein activity limit clinical translation. Despite this, the paradigm for the use of polymer particles in drug delivery has remained essentially unchanged for several decades. By taking advantage of the adsorption of protein therapeutics to poly(lactic-co-glycolic acid) (PLGA) nanoparticles, we demonstrate controlled release without encapsulation. In fact, we obtain identical, burst-free, extended-release profiles for three different protein therapeutics with and without encapsulation in PLGA nanoparticles embedded within a hydrogel. Using both positively and negatively charged proteins, we show that short-range electrostatic interactions between the proteins and the PLGA nanoparticles are the underlying mechanism for controlled release. Moreover, we demonstrate tunable release by modifying nanoparticle concentration, nanoparticle size, or environmental pH. These new insights obviate the need for encapsulation and offer promising, translatable strategies for a more effective delivery of therapeutic biomolecules.

Keywords: PLGA; affinity release; central nervous system; controlled release; drug delivery; hydrogel; protein.

Fig. 1. Two different PLGA np systems are compared for controlled protein release.

(A) Protein encapsulated in PLGA np dispersed in a hydrogel. (B) Protein and blank PLGA np dispersed in a hydrogel. For the latter, protein adsorbs to the PLGA np but is not encapsulated within them.

Fig. 2. Controlled sustained release of positively charged proteins from PLGA np does not require encapsulation.

(A to C) Three different proteins show nearly identical release profiles whether encapsulated within PLGA np or simply mixed with blank PLGA np in a hydrogel: (A) SDF (pI 10.9, molecular mass of 8 kD, 200 ng/release), (B) NT-3 (pI 9.5, molecular mass of 27 kD, 1000 ng/release), and (C) BDNF (pI 10.9, molecular mass of 27 kD, 300 ng/release). The cumulative percentage released is significantly greater for soluble proteins with no PLGA np compared to soluble proteins with blank PLGA np or proteins encapsulated in PLGA np at all time points (P < 0.05, n = 3 for all releases, mean ± SD plotted). Asterisks indicate significant differences between release of soluble protein with PLGA np and release of protein encapsulated in PLGA np (*P < 0.05, **P < 0.01, ***P < 0.001). Curves for soluble SDF with no PLGA np and SDF encapsulated in PLGA np were taken with permission (15). (D) EPO (pI ~4, molecular mass of 30 kD, 84 ng/release) shows no attenuated release when mixed into HAMC with PLGA np versus just HAMC alone (without PLGA np), indicating that the phenomena observed with SDF, NT-3, and BDNF are based on short-range electrostatic interactions between positively charged proteins and negatively charged PLGA (n = 3, mean ± SD plotted).

Fig. 3. Sustained release of BDNF from agarose containing PLGA np is disrupted by increased salt.

(A) BDNF shows the same delayed- and sustained-release profile from agarose containing PLGA np as from HAMC with PLGA np while diffusional release from agarose alone is still fast. (B) Almost all soluble BDNF is adsorbed to PLGA np after incubation, even at short times. (C) NaCl (0.5 M) completely disrupts the interaction of BDNF with PLGA np, resulting in purely diffusional release (P > 0.05 for all time points, n = 3 for all releases, mean ± SD plotted).

Fig. 4. Release of positively charged proteins from composite HAMC can be tuned by changing the available nanoparticle surface area.

(A) Cumulative percentage release of BDNF from composite HAMC is higher with 1000-nm-diameter PLGA np than with 300-nm-diameter PLGA np while keeping PLGA np mass constant. (B) (i) Release of NT-3 from composite HAMC with 0, 0.1, 0.5, 1, and 10 wt % PLGA np. Cumulative percentage of NT-3 released is significantly lower for 10 wt % PLGA np than for all other curves at t > 1 day (P < 0.05). Cumulative percentage of NT-3 released is significantly higher with 0 wt % PLGA np than with 0.1 wt % (t = 1 day, P < 0.05), 0.5 wt % (1 day ≤ t ≤ 3 days, P < 0.05), and 1 wt % (t > 3 hours, P < 0.05). (ii) Release of 0.5, 1, or 10 μg of NT-3 from composite HAMC with 10 wt % PLGA np. The concentration of NT-3 incorporated can be increased up to 20 times with virtually no change in release profile. Cumulative percentage of NT-3 released is significantly higher for 10 μg than for 1 and 0.5 μg at 10 and 14 days (P < 0.05) and is significantly lower for 0.5 μg than for 1 and 10 μg at 21 and 28 days (P < 0.05) (n = 3 for all releases, mean ± SD plotted).

Fig. 5. Release of positively charged proteins can be tuned by changing the environmental pH.

(A) Release of SDF from composite XMC into media at pH 3, pH 5, or pH 7. Cumulative percentage release of SDF is significantly greater at pH 3 than at pH 5 (t < 10 days, P < 0.01) and pH 7 (t > 0 days, P < 0.0001), likely because PLGA carboxylate anions are protonated to carboxylic acids, thereby reducing electrostatic interactions with positively charged proteins. (B) Release of BDNF from HAMC with PLGA np with or without encapsulated magnesium carbonate (MgCO₃). Release is delayed with encapsulated MgCO₃ (a basic salt), which neutralizes acidic degradation products and thereby maintains a higher/neutral local pH (n = 3 for all releases, mean ± SD plotted).

Fig. 6. Monte Carlo simulations agree well with experimental results.

Release of NT-3 from HAMC with 10 wt % PLGA np (Fig. 4B, i) was fitted using three-dimensional on-lattice Monte Carlo simulations. Simulations were then run for all other nanoparticle concentrations in Fig. 4B(i) using the same parameters. The combined reduced χ² value for all simulations was 3.9. Symbols represent data, whereas solid lines represent the simulation results.

Fig. 7. Adsorption may be rate-limiting for the release of positively charged proteins from PLGA np.

(i) Initially, the protein is fully adsorbed to the negatively charged nanoparticle surface. (ii) As the nanoparticle begins to degrade, acidic components build up and decrease the local pH. (iii) At a certain threshold, the nanoparticle surface becomes neutral, weakening the electrostatic interactions with the positively charged proteins and initiating release.