Formulation/Preparation of Functionalized Nanoparticles for In Vivo Targeted Drug Delivery

Summary Targeted cancer therapy allows the delivery of therapeutic agents to cancer cells without incurring undesirable side effects on the neighboring healthy tissues. Over the past decade, there has been an increasing interest in the development of advanced cancer therapeutics using targeted nanoparticles. Here we describe the preparation of drug-encapsulated nanoparticles formulated with biocompatible and biodegradable poly(D,L-lactic-co-glycolic acid)- block -poly(ethylene glycol) (PLGA-b -PEG) copolymer and surface functionalized with the A10 2-fluoropyrimidine ribonucleic acid aptamers that recognize the extracellular domain of prostate-specific membrane antigen (PSMA), a well-characterized antigen expressed on the surface of prostate cancer cells. We show that the self-assembled nanoparticles can selectively bind to PSMA-targeted prostate cancer cells in vitro and in vivo. This formulation method may contribute to the development of highly selective and effective cancer therapeutic and diagnostic devices.


Introduction
Nanomaterials have unique physicochemical properties, such as large surface area-to-mass ratios and high surface reactivity, which are different from bulk materials of the same composition. These unique physical properties allow the materials to interact with the human body on the molecular scale with a high degree of specificity. The application of nanotechnology in medicine, also known as nanomedicine, involves the use of precisely engineered nanomaterials for medical diagnosis and therapeutic treatments (1). One of the most exciting research topics in nanomedicine is targeted drug delivery. By combining molecular targeting capabilities and controlled drug release properties, targeted drug delivery offers the possibility of achieving precision-guided drug delivery to individual diseased cells with minimal side effects on neighboring healthy cells (2,3). as a model controlled release polymer system; and polyethylene glycol (PEG) as a model hydrophilic polymer with antibiofouling properties, to develop a proof-of-concept NP-Apt that is potentially suitable for selectively targeting PSMA PCa cells in vitro and in vivo. 4. PLGA-NHS was precipitated with 10 mL ethyl ether/methanol washing solvent to remove residual NHS and EDC.

5.
The precipitated PLGA-NHS was collected by centrifugation at 4,000 × g for 20 min.

6.
Washing and centrifugation (step 4 and 5) were repeated two times.

7.
The PLGA-NHS pellet was dried under vacuum for 30 min to remove the residual ether and methanol.

9.
The resulting PLGA-b-PEG block copolymer was precipitated with ether/methanol washing solvent and washed with the same solvent to remove unreacted PEG.

10.
The resulting purified PLGA-b-PEG block copolymer was dried under vacuum and used for NP preparation without further treatment (see Note 4).

11.
The composition of PLGA-b-PEG was characterized using a 400 MHz 1 H nuclear magnetic resonance (Bruker, Bill-erica, MA, USA). The nuclear magnetic resonance (NMR) characterization sample was prepared by dissolving 5 mg of the PLGA-b-PEG diblock copolymer in 1 mL of deuterated chloroform (CDCl 3 ). An example of a PLGA-b-PEG NMR spectrum is shown in Fig. 1.

2.
The PLGA-b-PEG and docetaxel mixture was added drop wise to three to five volumes of stirring water (see Note 5), giving a final polymer concentration of 3.3 mg/mL.

3.
The NPs were stirred for 2 h, and the remaining organic solvent was removed in a rotary evaporator at reduced pressure.

4.
The NPs were concentrated using Amicon ultracentrifugation at 4,000 × g for 15 min and washed with deionized water and reconstituted in PBS.

5.
The particle size and size distribution can be measured by dynamic light scattering (Brookhaven Instruments Corporation 90 plus particle sizer, 676-nm laser) at 25°C and at a scattering angle of 90° at a concentration of approximately 1 mg NP/mL water (see Note 6).

2.
The emulsion was then transferred to an aqueous solution of PVA (0.1%, w/v, 50 mL), and sonicated at 20 W for 1 min.

3.
The w/o/w emulsion formed was gently stirred at room temperature for 2 h or until the evaporation of the organic phase was complete.

4.
The nanoparticles were then recovered using Amicon ultracentrifugation as described in step 4 of Subheading 3.2.1 (see Note 6). 4 To achieve more efficient polymer conjugation, use a high-power vacuum pump to rapidly evaporate residual solvents in the polymer formulation. 5 To avoid nanoparticle aggregation, the acetonitrile:water volume should be greater than 2:1. 6 To maintain NP colloidal stability, always formulate NPs in pure water, then reconstitute NPs in PBS or other desired media.

8.
Where indicated, the number of nanoparticle aptamer biocon-jugates or control nanoparticles attached to individual LNCaP or PC3 cells was quantified by fluorescent microscopy under oil immersion at 100× magnification (see Note 8). A sample figure of targeted NP uptake by LNCaP and PC cells is shown in Fig. 3.

Efficacy of Tumor Reduction In Vivo
1. The NPs were traced by encapsulating docetaxel using the nanoprecipitation method explained in steps 1-5 of Subheading 3.2.1.

2.
The NP formulations were suspended in 200 μL PBS before administration.

LNCaP tumors were induced in 8-week-old balb/c nude mice (Charles River
Laboratories).

6.
Mice were injected subcutaneously in the right flank with 3 × 10 6 LNCaP cells suspended in a 1:1 mixture of media and Matrigel (BD Biosciences).

7.
Tumor-targeting studies were carried out after the mice developed ~100 mg tumors (see Note 10).

8.
Mice were randomly divided into different groups, minimizing tumor size variations between groups.

9.
Mice were anesthetized by intraperitoneal injection of Avertin (200 mg/kg body weight), and dosed with NP formulations via intratumoral injection.

10.
After dosing, the mice were monitored for weight and implanted tumor size change daily for 2 weeks and every 3 days thereafter.
11. If body weight loss (BWL) persisted beyond 20% of predosing weight, the animals were euthanized.

12.
The length and width of the tumors were measured by digital calipers, calculating tumor volume by the following formula: (width 2 × length)/2.

13.
Mice were monitored for a maximum of 109 days, until the tumor was completely regressed or until the tumor volume exceeded 800 mm 3 , for which the mice were euthanized for excessive tumor load.
14. For animals that were euthanized because of tumor load or BWL, the tumor size at the time of euthanasia was used for the purpose of mean tumor size calculation. 8 The fluorescent dyes encapsulated in NPs are released in a time-dependent manner. Always prepare a fresh batch of NPs for the in vitro release study to maximize the amount of dyes encapsulated in the NPs. 9 To obtain fast growing tumors, always reconstitute LNCaP cells in full growth media containing serum before mixing with Matrigel. 10 For maximum LNCaP growth, ensure all media are phenol free.
Initial volume of the tumors averaged 328 mm 3 . Tumor efficacy study results are shown in Fig. 4.