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stream. The results of the solubility screening show three different solvent compositions for six IPs. Pamoic acid (PA), sodium dodecyl sulfate (SDS), and sodium decyl sulfate (DS) dissolve in 50% dimethylsulfoxide (DMSO)/tetrahy› drofuran (THF); oleic acid (OA) and myristic acid (MA) dissolve in 50% methanol (MeOH)/THF; and sodium dode› cylbenzene sulfonate (DBS) dissolves in 20% water/THF. 

Nanocarrier Formation by Flash NanoPrecipitation (Ion Pairing of API:IP into PEG-Coated NPs) 

[0075] Flash NanoPrecipitation was used to self-assemble PEG-coated NCs containing an encapsulated API:IP com› plex. For the pre-ion paired method, particles were formed by rapid micro-mixing of hydrophobic-hydrophilic block› copolymer PCL-PEG and API:IP complex dissolved in organic solvent against deionized (DI) water through a confined impingement jet (CIJ) mixer. For the in situ ion pairing method, the organic stream containing PCL-PEG and anionic IP dissolved in organic solvent were rapidly micro-mixed in the CIJ mixer against the water stream containing cationic hydrophilic API dissolved in water. For both methods, the organic and water streams were mixed in a 1: 1 volume ratio and the resulting mixed streams were subsequently diluted tenfold into water. Concentrations of PCL-PEG stabilizer and hydrophilic API (gentamicin and polymyxin B) were kept constant, while IP concentrations were varied to form NCs, e.g., nanoparticles, with a range of drug (API):IP ratios. This ratio is the ratio of the molar concentration of cationic functional groups in the API, to the molar concentration of anionic functional groups in the IP. Exact compositions of the stabilizer block co-polymers, APis, and IPs used are given in Table 1 (above). 

## Nanocarrier Characterization 

[0076] Particle sizes and polydispersities were character› ized by dynamic light scattering (Malvern Zetasizer Nano, Malvern Instruments). Sizes were determined with back› scattering measurements and reported as Z-average and intensity-weighted distributions. The polydispersity index (PDI) reported herein is obtained from the Taylor series expansion of the autocorrelation function and is imple› mented into the Malvern Nanosizer data analysis software. A ratio of the second to the first moment is defined as the PDI, where values of 0.1 are generally obtained for monodisperse particles. 

[0077] Particles were diluted tenfold into two different collection buffers, deionized (DI) water and 1 x phosphate› buffered saline (PBS) for sizing measurements and NC, e.g., nanoparticle, stability assessments. PBS contains significant salt concentration, has an ionic strength of 163 mM, and mimics the ionic strength of serum. Incubation in PBS assesses the stability of the NC under conditions where ion exchange, e.g. with physiological charged species, might destabilize the NC ion-paired complex. NC stability was assessed by taking DLS measurements of the particles at 4 time points: t=0, 3 hr, 24 hr, and 72 hr. Stability measure› ments were conducted at room temperature. 

[0078] NC drug encapsulation efficiencies were deter› mined by separating encapsulated API from dissolved/un› encapsulated drugs in the NC sample through ultrafiltration across a 50 kDa membrane (Amicon Ultra, EMD Millipore) and determining the concentration of dissolved/unencapsu› lated API in ultrafiltration flow-through. Encapsulation effi› ciency is calculated as the concentration of API within the 

flow-through fraction divided by the concentration of API within the unseparated NC sample, and then subtracting this ratio from 1 (Eq. 1 ). 

[0079] Drug concentrations were determined with the bicinchoninic acid (BCA) assay (Spectramax i3x, Molecular Devices) with absorbance measurements at 562 nm, using a standard curve of the same drug (unencapsulated) as the reference. API fractional release was determined by dialyz› ing NC sample in 100-fold lxPBS. Aliquots were taken from within the dialysis bag at different time points: t=0, 1 day, 2 days, 3 days, 5 days, and 7 days. The drug concen› tration in each aliquot was determined by the BCA assay at 562 nm. The released fraction is the API concentration at each time point divided by the initial API concentration before dialysis (Eq. 2). 

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Example 1: Nanoparticles Encapsulating Gentamycin and Polymyxin B with Ion Pair Agents 

## Hydrophobic Ion Pair Screening 

[0080] For successful encapsulation through nanoprecipi› tation methods, API:IP complexes must rapidly precipitate when mixed with an antisolvent stream (in this case an aqueous solution). However, as shown in FIG. 1, the API:IP complex may be pre-formed, and then dissolved in a water miscible organic solvent phase, or the complex may be made in situ where the hydrophobic IP is dissolved in the organic phase with the stabilizing polymer, and the water-soluble API is introduced in the aqueous phase. The APis gentami› cin and polymyxin B (FIG. 2) are highly charged hydro› philic compounds, with water solubility of> 100 mg mL- 1 and >50 mg mL-$^{1 }$respectively, and with minimal solubility in polar organic solvents such as tetrahydrofuran (THF). 

[0081] We first investigated preformed API:IP complexes by the following sequence of studies: (1) determination of which ion-pairs (IPs) can be used to convert the highly water soluble APis gentamycin and polymyxin B into water› insoluble salt forms; and (2) production of salt forms that can be dissolved in highly nonpolar solvents containing THF and yield complexes that re-precipitate from THF when mixed with excess water. 

[0082] Several water-soluble counter ion salts IPs were chosen for this investigation to determine the physical properties of the counter ions required for successful com› plex formation (FIG. 3). A range of carboxylate and sul› fonate salt IPs whose acid forms have pKa ranging 5 to -1.9 were tested, as well as salts with a wide range of solubilities and Log P values as high as 7 .6 or as low as -4.92. API was dissolved at 5 mg mL-$^{1 }$in water in 1 x volume, and rapidly mixed with 1 x volume of dissolved IP to give a final mixture containing a 1: 1 API:IP charge ratio. Mixtures were visually observed to assess if precipitates rapidly formed. Out of the fifteen IPs tested with polymyxin B, eleven IPs resulted in the formation of precipitates; four IPs did not produce an observable precipitate (FIG. 4, Tables 1 and 2). Control experiments where API or IP alone were mixed did not result 

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