US 2020/0268679 Al  13  Aug. 27, 2020  [0098]  NC stability was assessed by measuring the size  distributions of the NCs when diluted into a closed volume  of water or PBS, all at room temperature. Although charac› terization of NPs in a closed volume does not capture the  sink conditions that would be present in vivo, behavior in  closed systems can shed insight on NP properties. When  paired with at least one charge equivalent of counterion for  the four counterions tested, nanoparticle size remained  mostly constant in water, despite minor ripening or swelling.  When diluted tenfold into PBS, however, particles exhibited  size changes over time, which are driven by ion exchange  between the anionic IP and the  c1- and  po$_{4}$ - 3  ions in PBS.  The ion exchange releases the bound API, which is soluble  in the external phase. In the case of pamoic acid IPs,  [0096]  NC formation using pre-formedAPI:IP complex is,  therefore, demonstrated. However, there are advantages to in  situ API:IP NC complex formation, since NC formation  could be accomplished in a single step. This single step  [0095]  No particles, only PCL-PEG micelles, were formed  when only stabilizer and API, or when only stabilizer and IP,  was used in the FNC process, with the exception of OA  (Table 1 ). Large aggregates were formed when only API and  IP was included during the FNC process, demonstrating that  the inclusion of all three stabilizer, API, and IP components  are simultaneously needed for NC formation. These results  demonstrate that while water-soluble APis cannot be for› mulated into NCs, hydrophobic API:IP complexes can be  formed into NCs with the water-precipitation process.  NC Stability  At a 1:1 IP to API charge ratio, polymyxin B was encapsu› lated with efficiency greater than 95% efficiency for all IPs  tested. At excess IP, high encapsulation efficiency was  retained, but at a 0.5:1 IP to API charge ratio, encapsulation  efficiency dropped significantly, to - 70% in the case of  sodium oleate. These results demonstrate that decreasing the  amounts of IP relative to API that is present can cause  decreased encapsulation efficiencies, by increasing the rela› tive amount of solubilized API that is not retained in the core  by ion pairing.  [0094]  The four IP candidates (OA, SDS, DBS, PA) that  passed the screening process were used to form NCs with the  FNP process. Gentamicin and polymyxin B were precipi› tated with IPs and dissolved into THF as described above,  and mixed with PCL-PEG dissolved in THF to yield an  organic THF stream containing both polymeric stabilizer  and API:IP complex. This stream was rapidly mixed against  water within a confined impingement jet to precipitate  API:IP complex in the presence of the amphiphilic PCL› PEG to form NCs. DLS analysis of the resultant samples  demonstrated that NCs 50-200 nm with narrow polydisper› sity were formed (FIGS. 6A and 6B, Table 1).  Flash NanoPrecipitation of API:IP Complexes  [0097]  To measure the encapsulation efficiency of poly› myxin B, free un-encapsulated drug was separated from that  which was encapsulated in NCs through ultrafiltration across  a 100 kDa membrane, and characterized with BCA analysis.  The encapsulation efficiency is based on the relative ratio of  free drug compared to the total amount of drug included in  the FNC system.  [0093]  For a system with a specific [ API:IPL, and Ks, the  fraction of initial API that precipitates increases as the initial  concentrations of API and IP increase, as shown in FIG.  SB.  The following conditions were used to generate FIG.  SB:  10- 2  M<[API]$_{0}$<l0 2  M; [API]$_{0}$=[IP]$_{0}$ ;  10- 6  M<[API:IPLa,  <l M; and Ks=l M- 1 . Below a critical startingAPI and IP  initial concentrations, no precipitates are formed. The com› plex can be driven to nearly complete complexation of API  by increasing the IP concentration, which corresponds to Le  Chatlier’s principle. However, this only occurs if individu› ally the species are soluble.  If  one species precipitates or  micellizes, then adding more of that species does not further  drive the reaction. In that case the individual ion concen› trations in Eq. 3 would be given by the solubility product of  the insoluble solid phase or by the CMC of the micellizing  species. In that case an additional set of solubility products  and mass balances would have to be added to the balances  given by Eqns. 3-12.  If  there are two solid phases that can  be formed, that is, an insoluble ion pair complex (API:IP)  and a solid phase of the IP, then NCs can be formed, but the  stoichiometry of the core of the NC will not be determined  by the stoichiometery of the ion pair, but by the molar ratio  of complex and insoluble IP. These results provide a model  to guide IP choice and precipitation reaction conditions for  NC processing, and the model supports the experimental  results observed.  would simplify processing. That is, although APis can be  pre-formed into hydrophobic API:IP complexes using batch  processes prior to FNP, a single continuous processing step  that can result in both API complexation and NP formation  can simplify production procedures and reduce production  costs. To test in situ complexation and NC formation and  assess if APis can be encapsulated in a single continuous  step by in situ complexation, polymyxin B dissolved in  water was impinged against organic solvent containing IP  and PCL-PEG in the FNP process. The IPs previously  identified as capable of forming NCs using pre-formed  complexes were studied using this method. Out of the IPs  assessed, all converted polymyxin B into hydrophobic forms  in situ during mixing to yield stable NCs with narrow size  distributions (FIG.  6C).  By comparing FIGS.  6B  and  6C,  where FIG.  6B  is for the pre-formed IP, and FIG.  6C  is the  in situ ion paired polymyxin, one sees that the NC sizes  produced by either process are essentially equivalent. Con› trol experiments, in which PCL-PEG and IP only, or PCL› PEG and polymyxin B only were used resulted in empty  micelles and not in NC formation. Control experiments  where IP and polymyxin B only were used resulted in the  formation of aggregations, together demonstrating that all  three components-stabilizer, API, and IP-are required for  in situ complexation and NC formation.  better prec1p1tate formers. For a system with a defined  [ API:IPLa,, and constant starting API and IP initial concen› trations, the fraction of initial API that precipitates increases  as the Ks of the complex decreases (FIG.  SA).  If  the complex  exhibits Ks above a critical value, no solids are formed.  These results highlight that IPs with strong counter ion  interactions with the API maximize complex formation› which is reflected in the experimental results observed,  where IPs with lower acid form  pKa  are more effective in  forming precipitates, even if the IP itself is less hydrophobic.  [Polymyxin  B] fm  Encapsulation Efficiency= 1- ----~› [PolymyxinB],0,01 