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).  [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  Example 1: Nanoparticles Encapsulating  Gentamycin and Polymyxin B with Ion Pair Agents  [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  Aug. 27, 2020  10  US 2020/0268679 Al  Nanocarrier Characterization  Hydrophobic Ion Pair Screening  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).  Encapsulation efficiency= 1 - [AP/]=, ;n  NC,  [API],otal  (1)  Released fraction=  [A!Pln solution  [AP/lnitial  (2) 