Real-Time Monitoring of ATP-Responsive Drug Release Using Mesoporous-Silica-Coated Multicolor Upconversion Nanoparticles

Abstract

Stimuli-responsive drug delivery vehicles have garnered immense interest in recent years due to unparalleled progress made in material science and nanomedicine. However, the development of stimuli-responsive devices with integrated real-time monitoring capabilities is still in its nascent stage because of the limitations of imaging modalities. In this paper, we describe the development of a polypeptide-wrapped mesoporous-silica-coated multicolor upconversion nanoparticle (UCNP@MSN) as an adenosine triphosphate (ATP)-responsive drug delivery system (DDS) for long-term tracking and real-time monitoring of drug release. Our UCNP@MSN with multiple emission peaks in UV-NIR wavelength range was functionalized with zinc-dipicolylamine analogue (TDPA-Zn(2+)) on its exterior surface and loaded with small-molecule drugs like chemotherapeutics in interior mesopores. The drugs remained entrapped within the UCNP-MSNs when the nanoparticles were wrapped with a compact branched polypeptide, poly(Asp-Lys)-b-Asp, because of multivalent interactions between Asp moieties present in the polypeptide and the TDPA-Zn(2+) complex present on the surface of UCNP-MSNs. This led to luminescence resonance energy transfer (LRET) from the UCNPs to the entrapped drugs, which typically have absorption in UV-visible range, ultimately resulting in quenching of UCNP emission in UV-visible range while retaining their strong NIR emission. Addition of ATP led to a competitive displacement of the surface bound polypeptide by ATP due to its higher affinity to TDPA-Zn(2+), which led to the release of the entrapped drugs and subsequent elimination of LRET. Monitoring of such ATP-triggered ratiometric changes in LRET allowed us to monitor the release of the entrapped drugs in real-time. Given these results, we envision that our proposed UCNP@MSN-polypeptide hybrid nanoparticle has great potential for stimuli-responsive drug delivery as well as for monitoring biochemical changes taking place in live cancer and stem cells.

Keywords: core−shell nanoparticles; luminescence resonance energy transfer (LRET); real-time monitoring; stimuli-responsive drug delivery; upconversion nanoparticle.

Figure 1

(A) Structural illustration of core–shell TDPA-Zn²⁺-UCNP@MSN. (B) TEM characterization of the UCNP core, β-NaYF₄: Yb³⁺/Tm³⁺ and (C) after growth of a NaYF₄:Yb³⁺/Er³⁺ shell. The inset of (B) shows the HR-TEM image of (100) crystallographic planes of the UCNP, confirming the hexagonal phase. (D,E) TEM characterization of the TDPA-Zn²⁺-UCNP@MSNs clearly revealed the core–shell nanoparticle with a mesoporous silica shell. (F) UV–vis absorption spectrum of the UCNP@MSNs (black line), TDPA-UCNP@MSNs (blue line) and TDPA-Zn²⁺-UCNP@MSNs (red line) in HEPES buffer solution (pH 7.4, 10 mM). The appearance of the characteristic absorption peak of TDPA at 270 and 290 nm indicated the successful conjugation of TDPA to the nanoparticle surface. The successful chelating of TDPA with Zn²⁺ to form metallic complex was confirmed by obtaining a red-shift of the peak at 290 to 312 nm, a similar spectral change observed in the formation of TDPA-Zn²⁺ complex (25 μM) in HEPES solution, as shown in the inset. (G) Upconversion luminescence of the UCNP (1 wt % in cyclohexane) and TDPA-Zn²⁺-UCNP@MSN (5 wt % in HEPES buffer, pH 7.4, 10 mM) with an excitation at 980 nm (80 W · cm⁻²).

Figure 2

(A) Model drug fluorescein release profile of polypeptide G2d2-wrapped TDPA-Zn²⁺-UCNP@MSNs in HEPES solution in the presence of different amount of ATP: a, 0; b, 1 mM; c, 5 mM; and d 10 mM. (B) Release of fluorescein from TDPA-Zn²⁺-UCNP@MSNs wrapped with different peptides in HEPEs solution in the presence of 5 mM ATP. a, G2d1; b, G2d2; c, G2d3; d, G2d4. (C) Release of fluorescein from G2d2-wrapped TDPA-Zn²⁺-UCNP@MSN in response to various biomolecules, indicates the specific binding of the polypeptide wrapped nanoparticles for ATP and its analogues. Nanoparticle is 0.2 wt % dispersion in 10 mM pH 7.4 HEPES buffer solution. Various biomolecules were added to the nanoparticles solution and incubated for 4 h to monitor the release of fluorescein, except that trypsin was incubated with nanoparticles solution at 37 °C for 24 h. (D) Fluorescein release from G2d2-wrapped TDPA-Zn²⁺-UCNP@MSNs at different pHs in the absence and presence of 5 mM ATP. pH was adjusted using 0.1 M NaOH and 0.1 M HCl solution.

Figure 3

(A) Schematic illustration of the working mechanism for the real-time monitoring of drug release using the proposed polypeptide-wrapped multicolor TDPA-Zn²⁺-UCNP@MSN. (B) Spectrum overlaps between the UV–vis absorption of anticancer drug DOX (30 μM in water) and CPT (20 M in methanol), and the emission of the core–shell UCNP@MSN (5 wt % in HEPES buffer). (C–D) Time dependent emission spectrum of the G2d2-wrapped TDPA-Zn²⁺-UCNP@MSNs load with DOX and CPT, respectively, in HEPES solution in the presence of 5 mM ATP. Concentration for nanoparticle is 0.2 wt %. The enhancement in the UV–vis emission indicated the progressive release of encapsulated anticancer drugs. (E) Linear relationships between the amount of drugs released and the ratiometric signal (R) of the UCNP, where the R for DOX is the ratio of I_472nm to I_656nm, and R for CPT is the ratio of I_365nm to I_656nm. The power density of 980 nm excitation was kept at 80 W · cm⁻². (F) Time-dependent release profiles for DOX and CPT monitored by using the ratiometric signal of UCNP.

Figure 4

Fluorescence microscopy images depicting the change in emission signal of UCNP and DOX in HeLa cells treated with polypeptide G2d2-wrapped TDPA-Zn²⁺-UCNP@MSNs at 3, 8, and 24 h after incubation.

Scheme 1

Schematic representation of the real-time monitoring of ATP-responsive drug release from polypeptide wrapped TDPA-Zn²⁺-UCNP@MSNs. Small molecule drugs were entrapped within the mesopores of the silica shell on the hybrid nanoparticle by branched polypeptide capping the pores through a multivalent interaction between the oligo-aspartate side chain in the polypeptide and the TDPA-Zn²⁺ complex on nanoparticles surface. The UV–vis emission from the multicolor UCNP under 980 nm excitation was quenched because of the LRET between the loaded drugs and the UCNP. Addition of small molecular nucleoside-polyphosphates such as ATP led to a competitive binding of ATP to the TDPA-Zn²⁺ complex, which displaced the surface bound compact polypeptide because of the high binding affinity of ATP to the metallic complex. The drug release was accompanied with an enhancement in the UV–vis emission of UCNP, which allows for real-time monitoring of the drug release via a ratiometric signal using the NIR emission of UCNP as an internal reference.

Scheme 2

Molecular structure of G2d, poly(Asp-Lys)-b-Asp.