pharmaceutics

Article
Preparation and Pharmacokinetic Characterization of an
Anti-Virulence Compound Nanosuspensions
Nan Wang 1,†,‡ , Feng Qi 1,†,§ , Xiaolong He 2, k , Honglan Shi 2 , David W. Anderson 3 , Hao Li 1, *,¶
and Hongmin Sun 4, *

1 Department of Mechanical and Aerospace Engineering, University of Missouri, Columbia, MO 65211, USA;
[email protected] (N.W.); [email protected] (F.Q.)
2 Department of Chemistry, Missouri University of Science and Technology, Rolla, MO 65409, USA;
[email protected] (X.H.); [email protected] (H.S.)
3 Ivogen Inc. (Subsidiary of Nanova, Inc.), Columbia, MO 65203, USA; [email protected]
4 Department of Medicine, Division of Cardiovascular Medicine, University of Missouri,
Columbia, MO 65212, USA
* Correspondence: [email protected] (H.L.); [email protected] (H.S.)
† These authors contributed equally to this work.
‡ Current Address: National Academic of Innovation Strategy, China Association for Science and Technology,
Beijing 100863, China.
§ Current Address: BeiGene (Beijing) Co., Ltd., Beijing 102206, China.
k Current Address: Frontage Laboratories, Inc. 700 Pennsylvania Drive, Exton, PA 19341, USA.
¶ Current Address: Ivogen Inc. (Subsidiary of Nanova, Inc.), Columbia, MO 65203, USA.



Abstract: Antibiotic resistance has become a worldwide public health threat due to the rapid evo-


lution and spread of antibiotic-resistant bacteria. CCG-211790 is a novel anti-virulence compound
Citation: Wang, N.; Qi, F.; He, X.;
that does not kill bacteria but could ameliorate human diseases by inhibiting expression of virulence
Shi, H.; Anderson, D.W.; Li, H.;
factors, thereby applying less selection pressure for antibiotic resistance. However, its potential
Sun, H. Preparation and
clinical use is restricted because of its poor aqueous solubility, resulting in formulation challenges.
Pharmacokinetic Characterization of
Nanosuspension technology is an effective way to circumvent this problem. Nanosuspensions of
an Anti-Virulence Compound
Nanosuspensions. Pharmaceutics 2021,
CCG-211790 with two different particle sizes, NanoA (315 ± 6 nm) and NanoB (915 ± 24 nm),
13, 1586. https://doi.org/10.3390/ were prepared using an antisolvent precipitation-ultrasonication method with Tween 80 as the sta-
pharmaceutics13101586 bilizer. Particle and pharmacokinetics (PK) of CCG-211790 nanosuspensions were characterized.
Both NanoA and NanoB demonstrated remarkable increases in dissolution rate compared with the
Academic Editors: Rossella Laurano bulk compound. The PK parameters of NanoA were comparable to those of CCG-211790 solution
and Monica Boffito formulation in intravenous or oral administration, suggesting that CCG-211790 nanosuspensions
with smaller particle size improved oral bioavailability and drug exposure compared to traditional
Received: 29 August 2021 formulations of drug candidates.
Accepted: 25 September 2021
Published: 29 September 2021
Keywords: biofilm; anti-virulence; wound infection; nanosuspension; pharmacokinetic

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1. Introduction
iations.
Antibiotic resistance has become a major public health threat worldwide [1,2]. Con-
ventional antibiotics select resistant bacterial pathogens by eliminating drug-sensitive
competitors while leaving drug-resistant bacteria to multiply [3]. The increasing occurrence
of antibiotic-resistant strains calls for new strategies that incur less selective pressure [4].
Copyright: © 2021 by the authors.
Anti-virulence agents that ameliorate human diseases by reducing the expression of viru-
Licensee MDPI, Basel, Switzerland.
lence factors rather than killing or inhibiting the growth of bacterial pathogens provide
This article is an open access article
distributed under the terms and
an alternative approach to treat infections by antibiotic resistant pathogens [5]. We have
conditions of the Creative Commons
identified a chemical series of low-molecular-weight compounds that target the expres-
Attribution (CC BY) license (https:// sion of virulence factors of Streptococcus pyogenes and Staphylococcus aureus [6–8]. CCG-
creativecommons.org/licenses/by/ 211790, with the IUPAC name 9-methoxy-3,5,5-trimethyl-2-((2,2,2-trifluoroethyl)thio)-5,6-
4.0/). dihydrobenzo[h]quinazolin-4(3H)-one, is one of the lead compounds [9]. Previous biologi-

Pharmaceutics 2021, 13, 1586. https://doi.org/10.3390/pharmaceutics13101586 https://www.mdpi.com/journal/pharmaceutics

Pharmaceutics 2021, 13, 1586 2 of 19

cal studies demonstrated that CCG-211790 inhibited the biofilm formation of methicillin-
resistant Staphylococcus aureus (MRSA) [9], a biofilm forming nosocomial pathogen and one
of the most notorious multi-drug resistant bacteria that causes skin and soft tissue infections
(SSTIs) [10–14]. Bacterial biofilm formation and drug resistance make the infected wounds
recalcitrant to antibiotic treatment and could lead to systemic life-threatening infections and
sepsis [14]. Biofilm formation is a key pathology of non-healing wounds [15,16]. Bacteria in
biofilms are much more tolerant to antimicrobial agents and disinfectants [17–19], as well as
host defense mechanisms [20,21]. Potent antibiotics or their combinations are usually used
to treat complicated SSTIs caused by biofilm forming and multi-drug resistant bacteria,
but their use may be associated with life-threatening toxicities such as nephrotoxicity and
hepatotoxicity [22–24]. Thus there is a great unmet clinical need for novel anti-biofilm
agents to treat infections associated with chronical wounds [14].
CCG-211790 and its analogs inhibit MRSA virulence and biofilm formation, making
them attractive candidates for monotherapy or complementing current antibiotics for
treating wound infections [6,9]. However, the clinical use of CCG-211790 is restricted by
its low aqueous solubility (less than 50 ng/mL), leading to formulation challenges such
as erratic absorption and poor oral bioavailability for oral formulations [25]. A topical
formulation of CCG-211790 was developed for application in wound treatment [9], yet
there are many challenges to treating skin infections locally. Wet wound environment and
enzymes within the wound tissue reduce the efficacy of the topically applied drugs [26].
As a result, it is necessary to further improve the pharmacokinetic properties of this novel
anti-biofilm compound to enhance its potential for treating wound infections. Absorption
of orally administrated solid dosage forms of drugs into the systemic circulation involves
three factors. Dosage form disintegration, drug dissolution, and drug permeation across
intestinal cell membranes into the systemic circulation all affect drug absorption [27–30].
For poorly water-soluble drugs, especially the BCS II compounds, drug absorption is
often limited by the drug dissolution rate from the dosage forms (kd <<ka ). The maximum
drug plasma concentration (Cmax ) and time to reach the Cmax for this type of poorly
water-soluble drug is dictated by the dissolution rate of the drug from the dosage form.
Additionally, the fraction of drug absorbed will be affected by the drug dissolution rate if
the time required for complete dissolution is longer than the transit time of the dosage form
at the drug absorptive sites. These properties affect the overall bioavailability of drugs and
their overall effectiveness [27].
In this study, nanosuspension technology that reduces the particle size of drug
crystals to submicron scale was employed for the development of CCG-211790 formula-
tions. Nanosuspension is defined as the colloidal dispersion of nanosized drug particles
(100–1000 nm) in an aqueous vehicle such as water, aqueous solution or non-aqueous
media stabilized with surfactants or polymeric stabilizers [28]. Nanosuspension technology
is a promising approach for addressing poor-solubility problems and has many advantages
including: (1) enhanced dissolution rate and oral bioavailability compared with raw drugs
or coarse suspensions [31,32]; (2) high drug payload (e.g., 20–30% concentrated nanosus-
pensions produced via wet milling method, and up to 90% nanocrystal powder-loaded
tablets) is available with benefits to high-dosing situations [33,34]; (3) nanoparticles contain
almost 100% pure drug with low content of irritant excipients (e.g., surfactant and/or co-
solvents) that reduces excipient-induced toxicity and improves drug safety [35]; (4) diverse
dosage forms such as tablets, pellets, nanosuspensions, suppositories and hydrogels are
available giving flexibility for various administration routes such as intravenous (IV) [36],
intramuscular [37], oral [38], ocular [39], and nebulization [40].
In general, nanosuspensions can be produced either by bottom-up approaches consist-
ing of dissolved drug molecules precipitated or crystallized from a supersaturated solution
forming solid nanoparticles [41,42], or by top-down approaches where large particles are
reduced into nanosized particles via comminution processes [43,44]. In this study, two
CCG-211790 nanosuspension formulations with different particle sizes and morpholo-
gies, termed as NanoA (with z-average of 315 ± 6 nm) and NanoB (with z-average of
Pharmaceutics 2021, 13, 1586 3 of 19

915 ± 24 nm), respectively, were prepared using antisolvent precipitation-ultrasonication

method, which is a bottom-up approach. Particle size, morphology, stability, in vitro disso-
lution profiles, and in vivo pharmacokinetics (PK) of nanosuspensions were investigated.

2. Materials and Methods

2.1. Materials
The synthesis of CCG-211790 has been described previously [9]. Tween® 80 (polyoxyethylene-
20-sorbitan monooleate), polyethylene glycol 400 (PEG 400) and dimethyl sulfoxide (DMSO)
were purchased from Sigma-Aldrich (Saint Louis, MO, USA). Propylene glycol was pur-
chased from MP Biomedicals LLC (Irvine, CA, USA). Acetonitrile and sodium dodecyl
sulfate (SDS) were purchased from Fisher Scientific (Pittsburgh, PA, USA). Fractionated co-
conut oil was purchased from Piping Rock Health Products, LLC (Ronkonkoma, NY, USA).
Ethanol was purchased from Decon Laboratories Inc. (Detroit, MI, USA). Liquid coconut
oil was purchased from Piping Rock Health Products, LLC (Ronkonkoma, NY, USA) and
used as received. Deionized water was supplied from a Culligan reverse osmosis filtra-
tion system (Rosemont, IL, USA). All chemicals except coconut oil were of analytical or
high-performance liquid chromatography (HPLC) grade.

2.2. Preparation of CCG-211790 Nanosuspensions

CCG-211790 nanosuspensions with two different particle sizes were prepared by an
antisolvent precipitation-ultrasonication method and were named NanoA (315 ± 6 nm)
and NanoB (915 ± 24 nm), depending on the hydrodynamic particle size obtained by
dynamic light scattering (DLS). Briefly, NanoA was obtained by injecting 0.5 mL of CCG-
211790 solution (15 mg/mL CCG-211790 in DMSO) into 5 mL of Tween 80 solution (0.005%
w/v in water) under magnetic stirring (PC-420D stirring hot plate, Corning Inc., Corning,
NY, USA) at 1000 rpm, continuing with intense sonication for 10 min in an ultrasonic
bath (Bransonic B221, Emerson Electric, St. Louis, MO, USA) filled with 600 mL of water.
During this process, mechanical stirring at 600 rpm was applied by an overhead mixer
(OS40-S, Scilogex LLC, Rocky Hill, CT, USA). Similarly, NanoB was obtained by injecting
0.5 mL of CCG-211790 solution (15 mg/mL CCG-211790 in DMSO) into 5 mL of Tween
80 solution (0.02% w/v in water) under magnetic stirring at 500 rpm, continuing with
intense sonication and mechanical stirring at 400 rpm for 10 min in the ultrasonic bath
filled with 220 mL water. DMSO was then removed from suspensions by dialysis (Fisher
brand regenerated cellulose membrane with 12,000 to 14,000 MW cutoff) against water.
CCG-211790 concentration was adjusted to 1 mg/mL with water prior to use.

2.3. Dynamic Light Scattering (DLS)

The mean hydrodynamic particle size (z-average or zavg ), polydispersity index (PDI)
and zeta potential of CCG-211790 suspensions were determined by the Zetasizer Nano ZS
(Malvern Panalytical, Malvern, United Kingdom). The measurements of hydrodynamic
particle size and PDI were performed in deionized water at 25 ◦ C [45]. PDI is a parameter
evaluating the width of particle size distribution. Mean values and standard deviations
were calculated.

2.4. Scanning Electron Microscopy

The morphologies of freeze-dried CCG-211790 nanosuspensions and CCG-211790
bulk powder were observed using a scanning electron microscope (SEM) (FEI Company,
Hillsboro, OR, USA). SEM samples of nanosuspensions were prepared by freeze-drying
CCG-211790 nanosuspensions in a FreeZone 1 Liter Benchtop Freeze Dryer (Labconco
Corporation, Kansas City, MO, USA) immediately after preparation. Briefly, suspensions
(0.5 mL) were pre-frozen at −50 ◦ C for 2 h and then were freeze-dried at −50 ◦ C and
0.015 mbar for 48 h to yield dry powders. CCG-211790 bulk powder was used as received
following synthesis. Each sample was fixed on an aluminum SEM stage with a conductive
double-sided carbon tape.
Pharmaceutics 2021, 13, 1586 4 of 19

2.5. High-Performance Liquid Chromatography (HPLC) Analysis of Nanosuspensions

The concentrations of CCG-211790 nanosuspensions were measured using an Agilent
series 1100 HPLC system (Agilent Technologies, Santa Clara, CA, USA) equipped with a
diode array detector. Sample separation was carried out with a Kinetex C18 column
(75 mm × 3 mm, particle size 2.6 µm, Phenomenex, Torrance, CA, USA). The mobile
phase was acetonitrile-water (50:50, v/v), flow rate was set to 0.5 mL/min, and column
temperature to 35 ◦ C. The injection volume was 30 µL. The absorption wavelength was
set at 255 nm. CCG-211790 nanoparticles were dissolved in the mobile phase for HPLC
analysis. Due to the fact that the analyte concentration was high and no other matrix was
present in the sample, the HPLC method performed very well with excellent calibration
linearity, reproducibility, and spike recovery. During sample analysis, ongoing quality
controls were performed including a reagent blank, a duplicate sample, and spike recovery
samples for every 10 samples analyzed.

2.6. Stability of CCG-211790 Nanosuspensions

The representative batch of NanoA and NanoB nanosuspensions were stored at 4 ◦ C
or room temperature. Z-average, PDI and zeta potential were monitored by DLS over
a period of six weeks. Due to the fact that NanoA was produced in 0.005% Tween 80
solution while NanoB was produced in 0.02% Tween 80 solution, both NanoA and NanoB
nanosuspensions (0.2 mL) were diluted with 0.02% Tween 80 solution (2 mL) prior to each
measurement to maintain consistent medium vehicle.

2.7. In Vitro Dissolution Study

The in vitro dissolution profiles of CCG-211790 bulk powder, NanoA and NanoB
suspensions were examined. SDS is one of the most frequently used artificial surfactants
in dissolution test to establish sink conditions for water-insoluble drugs. Therefore, a
SDS solution (0.5% w/v in water) was used as the dissolution medium in this study. In
each dissolution test, suspension or powder equivalent to 4 mg of CCG-211790 were
directly dispersed in 500 mL dissolution medium under magnetic stirring at 75 rpm. The
temperature of the dissolution medium was maintained at 37 ± 1 ◦ C in a water bath.
Sample aliquots (3 mL) were withdrawn at predetermined time points (5, 10, 20, 30, 45, and
60 min) and immediately replaced with 3 mL of fresh dissolution medium to readjust the
volume to 500 mL. Undissolved particles were separated by centrifugation at 10,000× g
for 5 min and the supernatant was collected for HPLC analysis. All experiments were run
in triplicate.

2.8. Pilot PK Study of CCG-211790 Powder Suspension

The pilot PK studies of CCG-211790 powder suspension formulations via intravenous
administration and oral delivery were outsourced to Gateway Pharmacology Laboratories
(Chesterfield, MO, USA). Four male Sprague-Dawley rats (180–200 g) were purchased
from Harlan Laboratories (Indianapolis, IN, USA). All animals were acclimatized at a
temperature of 22 ± 4 ◦ C and a relative humidity of 30–70% under a 12 h light/dark
condition (lights on at 6:00 AM and off at 6:00 PM). All animals were allowed free access to
standard rodent chow (PicoLab Rodent Diet 20) and water.
A solution formulation of CCG-211790 to a concentration of 0.5 mg/mL was freshly
prepared prior to dosing by mixing 30 µL stock solution (50 mg/mL in DMSO) into 2970 µL
0.9% saline. Two rats received the solution formulation 2.5 mg/kg CCG-211790 via tail vein
injection at a dosing volume of 5 mL/kg with blood samples collected at 5, 15, and 30 min;
1, 3 (via retro-orbital bleeds) and 6 (via cardiac puncture) hours post-dose. Another two
rats received 5 mg/kg CCG-211790 bulk powder dispersed in 0.5% w/v of carboxymethyl
cellulose (CMC) solution via oral gavage at a dosing volume of 10 mL/kg. Blood samples
were collected at 0.5, 1, 2, 4, 6 (via retro-orbital bleeds), and 8 (via cardiac puncture) hours
post-dose. Plasma samples were analyzed by HPLC-MS/MS method. Analysis was per-
formed using a Shimadzu HPLC system (Shimadzu Corporation, Columbia, MD, USA)
Pharmaceutics 2021, 13, 1586 5 of 19

equipped with a 4000Q Trap tandem mass spectrometer (AB Sciex, Foster City, CA, USA).
The method was developed by tuning the instruments for the test article based on the
molecular weight of the compound, following an industry standard fit-for-purpose ap-
proach/assay for pharmacokinetic/pharmacology studies where concentration versus time
data were beneficial in understanding the exposures. A Kinetex C18 (2.1 × 50 mm, particle
size 2.6 µm, Phenomenex, Torrance, CA, USA) was used for separation. Samples were
eluted with a flow rate set to 0.4 mL/min under a gradient elution program with eluent
A (ultra-pure water with 0.1% (v/v) formic acid) and eluent B (acetonitrile with 0.1% (v/v)
formic acid).

2.9. PK Studies of Nanosuspensions

PK studies of nanosuspensions via intravenous administration and oral administration
were performed in male Sprague-Dawley rats (450 ± 32 g and 425 ± 19 g, respectively).
All animals were purchased from Charles River Laboratories (Wilmington, MA, USA). The
animals were acclimatized at a temperature of 25 ± 2 ◦ C and a relative humidity of 75 ± 5%
under natural light/dark condition for one week. The animals were kept under fasting
overnight prior to the experiments. All experimental procedures were approved by the
Animal Care and Use Committee (ACUC) of University of Missouri (Columbia, MO, USA).
For IV PK studies, Sprague-Dawley rats were randomly divided into three groups
with six rats in each group. A solution formulation of CCG-211790 dissolved in a solution of
ethanol (10%), propylene glycol (25%), PEG 400 (35%) and water (30%) to a concentration of
1 mg/mL, termed as PEG/PG solution, was prepared as the control. The PEG/PG solution,
NanoA and NanoB suspensions were administrated to the three groups respectively at a
dose of 2.5 mg/kg via bolus tail vein injection. Blood samples were collected in EDTA-
coated tubes via femoral vein bleeding at predetermined time points (5, 15, and 30 min;
1 h, 2 h, and 4 h). Plasma was separated from the blood by centrifugation at 1000× g for
10 min and stored at −80 °C until analysis.
For oral PK studies, Sprague-Dawley rats were randomly divided into three groups
with six rats in each group. A coconut oil solution formulation of CCG-211790 dissolved
in fractionated coconut oil to a concentration of 1 mg/mL was prepared as the control.
The coconut oil solution and NanoA and NanoB suspensions were administrated by oral
gavage (10 mL/kg) at a dose of 5 mg/kg, respectively. Blood samples were collected
in EDTA-coated tubes via femoral vein bleeding at predetermined time points (0.5, 1,
2, 4, 6, and 8 h post-dose). Plasma was separated from the blood by centrifugation at
1000× g for 10 min and stored at −80 ◦ C until analysis. Plasma samples were analyzed by
UPLC-MS/MS method.

2.10. UPLC-MS/MS Analysis of Plasma Samples

The concentration of CCG-211790 in plasma is low and a more sensitive UPLC-MS/MS
method was developed to detect its concentration in plasma samples. A procedural cal-
ibration protocol was used for calibration standard preparation using a control plasma
that did not contain CCG-211790 and internal standard. Different concentrations of cal-
ibration standard were added into the control plasma and processed through the same
procedure as the plasma sample preparation. For standard and sample preparation, 30 µL
plasma was mixed with 120 µL acetonitrile containing an internal standard tolbutamide
followed by 30 min wait to completely precipitate the protein in the plasma, which was
then centrifuged at 10,000× g for 10 min. The supernatant (100 µL) was transferred to an
autosampler vial for UPLC-MS/MS analysis. UPLC-MS/MS analysis was performed using
a Shimadzu UPLC system (Shimadzu Corporation, Columbia, MD, USA) equipped with a
4000Q Trap tandem mass spectrometer (AB Sciex, Foster City, CA, USA). A Gemini 3u C18
column (50 mm × 2 mm, particle size 3 µm, Phenomenex, Torrance, CA, USA) was used
for separation. Samples were eluted with a flow rate set to 0.8 mL/min under a gradient
elution program with eluent A (ultra-pure water with 0.1% (v/v) formic acid) and eluent B
(acetonitrile with 0.1% (v/v) formic acid). The gradient was set as follows: 20 % eluent B
Pharmaceutics 2021, 13, 1586 6 of 19

maintained for 1 min followed by linear increase to 100% over 2 min and maintained at
100% eluent B for 3 min. The total runtime was 6 min. The sample injection volume was
10 µL. Column temperature was set to 30 ◦ C. The 4000Q Trap spectrometer was operated
under positive electrospray ionization (+ESI) mode and using multiple reaction monitoring
(MRM) for quantitation. The quantification ion pair was 385.088 > 156 and confirmation
ion pair was 385.088 > 73.1 for CCG-211790. The quantification ion pair used for internal
standard was 271.108 > 90.9 and confirmation ion pair was 271.108 > 74. All the other
parameters were optimized for the most sensitive ion transition for quantification. The
method was validated prior to plasma sample analysis. The validation conditions included
reproducibility, calibration linearity, detection limit, and spike recovery. The method
quantification detection limit was 0.5 ng/mL. The calibration showed very good linearity
(R2 > 0.99). Strict ongoing quality control tests were performed during all the sample
analyses, including a quality control standard check, a reagent blank, a spike recovery, and
a duplicate sample for every 10 samples analyzed to certify precisions and accuracies of
the analyses.

2.11. Statistics
PK parameters were analyzed via non-compartmental analysis using PK solver 2.0
software, which is a freely available menu-driven add-in program for Microsoft Excel, for
PK and pharmacodynamic (PD) data analysis (China Pharmaceutical University, Nanjing,
China) [46]. Statistical differences were estimated via Student’s t-test with p < 0.05 as
significant.

3. Results
3.1. Particle Size and Morphology
The hydrodynamic particle size and the z-average, of NanoA and NanoB suspensions
were determined by DLS after the dialysis. It is worth noting that the hydrodynamic particle
size is an apparent value equivalent to the diameter of a sphere having the same average
diffusion coefficient as the particles being measured [47]. In this study, the preparation of
NanoA samples (n = 21) demonstrated good reproducibility, while NanoB samples (n = 9)
displayed wider particle size distribution. In general, the z-average values of NanoA
ranged from 300–400 nm with a mean value of 317 ± 20 nm. Similarly, the z-average
values of NanoB samples ranged from 680–950 nm with a mean value of 801 ± 110 nm.
One representative batch of NanoA (315 ± 6 nm) and one representative batch of NanoB
(915 ± 24 nm) were selected for the following stability study (Figure 1).
The morphologies of CCG-211790 bulk powder, NanoA and NanoB freeze-dried
powders were examined by SEM. The majority of particles in NanoA were small particles
with dimensions from 200–400 nm, while a few larger plate-like particles also existed with
lengths typically from 500–1100 nm and widths less than 400 nm (Figure 2A,B). In contrast,
plate-like and tube-like particles were produced in NanoB with lengths mostly around
0.5–3 µm, widths at the submicron-scale and thicknesses down to a hundred nanometers
(Figure 2C,D). CCG-211790 bulk powder was more heterogeneous and consisted of a large
number of relatively small particles with lengths up to twenty micrometers and diameters
of a few micrometers. In addition, a few relatively large particles with lengths over fifty
micrometers and diameters over twenty micrometers were observed, demonstrating the
heterogeneity of the bulk powder (Figure 2E,F).
Pharmaceutics 2021, 13, 1586 7 of 19

Figure 1. Hydrodynamic particle sizes of the representative batch of NanoA and NanoB suspension
after the preparation obtained by DLS.

Figure 2. SEM images of NanoA (A,B), NanoB (C,D), and CCG-211790 bulk powder (E,F).
Pharmaceutics 2021, 13, 1586 8 of 19

3.2. Storage Stability of Nanosuspensions

NanoA and NanoB suspensions were stored at 4 ◦ C and room temperature, z-average,
PDI and zeta potential were monitored by DLS for six weeks. Although DLS does not
produce results directly related to the size of high aspect-ratio particles, it is still useful to
roughly track the change of the particle size over time [48].
At both 4 ◦ C and room temperature, there were no significant differences of z-average
(315 ± 6 nm vs. 315 ± 3 nm, 315 ± 6 nm vs. 319 ± 5 nm), and PDI (0.18 ± 0.03 vs.
0.21 ± 0.01, 0.18 ± 0.03 vs. 0.20 ± 0.03) for NanoA suspension after 6 weeks, with similar
results found for NanoB. Overall, both NanoA and NanoB were relatively stable within
6 weeks, (Tables 1 and 2), probably because a minimum zeta potential of ±30 mV was
sufficient to provide electrostatic barrier against particle-particle aggregation [49,50].

Table 1. Stabilization of NanoA suspension storing at 4 ◦ C or room temperature (RT) over six weeks.

Duration Z-Average (nm) Polydispersity Index Zeta Potential (mV)

(Week) 4 ◦C RT 4 ◦C RT 4 ◦C RT
0 315 ± 6 315 ± 6 0.18 ± 0.03 0.18 ± 0.03 −42.3 ± 4.2 −42.3 ± 4.2
1 311 ± 1 310 ± 3 0.21 ± 0.01 0.22 ± 0.01 −49.3 ± 0.5 −48.4 ± 0.3
2 309 ± 3 316 ± 7 0.20 ± 0.03 0.20 ± 0.03 −49.4 ± 1.3 −49.5 ± 0.9
4 314 ± 2 322 ± 6 0.21 ± 0.01 0.23 ± 0.01 −46.1 ± 0.4 −52.3 ± 0.7
6 315 ± 3 319 ± 5 0.21 ± 0.01 0.20 ± 0.03 −45.4 ± 0.4 −51.3 ± 0.3
Each sample was measured in triplicate with mean values and standard deviations. No statistically significant
difference was observed in comparison to their initial values (p > 0.05).

Table 2. Stabilization of NanoB suspension storing at 4 ◦ C or room temperature over six weeks.

Duration Z-Average (nm) Polydispersity Index Zeta Potential (mV)

(Week) 4 ◦C RT 4 ◦C RT 4 ◦C RT
0 915 ± 24 915 ± 24 0.52 ± 0.03 0.52 ± 0.03 −42.2 ± 0.2 −42.2 ± 0.2
1 900 ± 99 947 ± 60 0.58 ± 0.12 0.62 ± 0.02 * −49.5 ± 0.5 * −50.4 ± 0.7 *
2 911 ± 37 912 ± 66 0.61 ± 0.01 * 0.62 ± 0.07 −44.9 ± 0.6 * −49.1 ± 0.7 *
4 966 ± 89 878 ± 17 0.59 ± 0.03 0.54 ± 0.04 −47.7 ± 1.0 * −50.2 ± 0.3 *
6 856 ± 17 * 891 ± 24 0.57 ± 0.05 0.60 ± 0.05 −46.4 ± 1.4 * −50.0 ± 1.5 *
Each sample was measured in triplicate with mean values and standard deviations. * p < 0.05, statistically
significant difference was observed in comparison to their initial values.

3.3. In Vitro Dissolution Studies

Dissolution profiles (Figure 3) showed significant increases in dissolution rates for
NanoA and NanoB suspensions compared to CCG-211790 bulk powder. The drug releases
for NanoA and NanoB suspensions were 99% at 5 min whereas bulk powder CCG-221790
release was 19.6% at 5 min. Bulk powder release was slow, reaching a maximum of 64.3%
after 60 min.
Pharmaceutics 2021, 13, 1586 9 of 19

Figure 3. In Vitro dissolution profiles of CCG-211790 bulk powder and NanoA and NanoB sus-
pensions in 0.5% SDS solution (n = 3). There were statistically significant differences between the
dissolution rates of NanoA and NanoB and the dissolution rate of CCG-211790 bulk powder (p < 0.05),
while there was no statistical difference between NanoA and NanoB (p > 0.05).

3.4. Pilot PK Studies

The CCG-211790 plasma concentration-time curves and the PK parameters for pilot
study are shown in Figure 4, Tables 3 and 4 respectively. Bioavailability (F) is one of the
principal pharmacokinetic properties of drugs defined as the fraction of an administered
dose of unchanged drug that reaches systemic circulation [51]. The bioavailability of a
drug given by the intravenous route is considered 100% (F = 1), and usually less than one
if given by other routes; bioavailability close to 100% indicates complete absorption of the
drug, which is desirable in the development of extra-vascular or oral formulations [52]. The
AUC · Dose
po IV
bioavailability of an oral formulation is given by F = AUCIV · Dose PO × 100%, where AUC is
the area under the concentration-time curve, and DoseIV and DosePO were 2.5 mg/kg and
5 mg/kg, respectively, for the pilot PK study.

Figure 4. Plasma concentration–time curve for pilot study of CCG-211790 powder suspension (n = 2) and a DMSO/saline
solution (n = 2) either following oral administration or intravenous administration in Sprague–Dawley rats at a dose of
5 mg/kg or 2.5 mg/kg, respectively.
Pharmaceutics 2021, 13, 1586 10 of 19

Table 3. Pharmacokinetic parameters after intravenous administration of the DMSO/saline solution

of CCG-211790 at a dose of 2.5 mg/kg in male Sprague–Dawley rats (n = 2).

PK Parameters Pilot Study for IV

Cmax (mg L−1 ) 1.21 ± 0.15
AUC0−∞ (mg L−1 h) 0.67 ± 0.18
MRT0−∞ (h) 0.95 ± 0.07
t1/2β (h) 1.44 ± 0.15
Vz (L kg−1 ) 8.07 ± 2.92
CL (L h−1 kg−1 ) 3.85 ± 1.01
AUC: area under the concentration–time curve; Cmax : maximal concentration; MRT: mean residence time; t1/2β :
terminal half-life; Vz : volume of distribution during terminal phase; CL: clearance.

Table 4. Pharmacokinetic parameters of CCG-211790 powder suspension following oral administra-

tion at a dose of 5 mg/kg in male Sprague–Dawley rats (n = 2).

PK Parameters Pilot Study for PO

Cmax (mg L−1 ) 0.04 ± 0.01
Tmax (h) 1.50 ± 0.71
AUC0−∞ (mg L−1 h) 0.18 ± 0.03
MRT0−∞ (h) 4.75 ± 1.20
t1/2β (h) 3.88 ± 1.68
Vz /F (L kg−1 ) 168 ± 101
CL/F (L h−1 kg−1 ) 28.8 ± 5.6
FIV 13.4%
Tmax : time for Cmax ; Vz /F: apparent volume of distribution during terminal phase after non-intravenous ad-
ministration; CL/F: apparent total clearance of the drug from plasma after oral administration; FIV : absolute
bioavailability in regard to DMSO/saline solution following intravenous administration.

The bulk compound had poor oral bioavailability at 13.4%, likely due to poor aqueous
solubility of the compound [25].

3.5. PK Studies
The CCG-211790 plasma concentration-time curves and the PK parameters of PEG/PG
solution (control), NanoA and NanoB suspensions following intravenous administration in
Sprague-Dawley rats at a dose of 2.5 mg/kg are shown in Figure 5 and Table 5, respectively.

Figure 5. Plasma concentration–time curve of PEG/PG solution and NanoA and NanoB suspensions
after intravenous administration in Sprague–Dawley rats at a dose of 2.5 mg/kg (n = 6).
Pharmaceutics 2021, 13, 1586 11 of 19

Table 5. PK parameters after intravenous administration of PEG/PG solution and NanoA and NanoB
suspensions at a dose of 2.5 mg/kg in male Sprague–Dawley rats (n = 6).

PK Parameters PEG/PG NanoA NanoB

Cmax (mg L−1 ) 1.63 ± 0.25 1.31 ± 0.55 1.07 ± 0.44 *
AUC0−∞ (mg L−1 h) 1.11 ± 0.14 0.64 ± 0.19 * 0.49 ± 0.12 *
MRT0−∞ (h) 0.98 ± 0.17 0.90 ± 0.14 0.77 ± 0.21
t1/2β (h) 1.13 ± 0.27 1.08 ± 0.18 1.06 ± 0.27
Vz (L kg−1 ) 3.66 ± 0.93 6.44 ± 1.84 * 8.37 ± 3.29 *
CL (L h−1 kg−1 ) 2.28 ± 0.27 4.21 ± 1.28 * 5.28 ± 1.01 *
* p < 0.05, statistically significant difference compared with PEG/PG solution.

NanoA and NanoB showed trends toward lower Cmax (1.31 ± 0.55 mg L−1 ,
1.07 ± 0.44 mg L−1 ), as compared to PEG/PG solution (1.63 ± 0.25 mg L−1 ). The clear-
ance (CL) of NanoA (4.21 ± 1.28 L h−1 kg−1 ) and NanoB (5.28 ± 1.01 L h−1 kg−1 ) were
higher than that of PEG/PG solution (2.28 ± 0.27 L h−1 kg−1 ). The AUC of NanoA
(0.64 ± 0.19 mg L−1 h) and NanoB (0.49 ± 0.12 mg L−1 h) were lower than that of PEG/PG
solution (1.11 ± 0.14 mg L−1 h). The Vz s of the NanoA (6.44 ± 1.84 L kg−1 ) and NanoB
(8.37 ± 3.29 L kg−1 ) were higher than PEG/PG solution (3.66 ± 0.93 L kg−1 ).
The CCG-211790 plasma concentration-time curves and the PK parameters of coconut
oil solution, NanoA and NanoB suspensions following oral administration in Sprague-
Dawley rats at a dose of 5 mg/kg are shown in Figure 6 and Table 6, respectively.

Figure 6. Plasma concentration–time curve of the coconut oil solution (n = 6) and NanoA and NanoB
suspensions (n = 6) after oral administration in Sprague–Dawley rats at a dose of 5 mg/kg.

The Cmax values for NanoA (0.13 ± 0.05 mg L−1 ), NanoB (0.12 ± 0.03 mg L−1 ), and
coconut oil solution (0.12 ± 0.07 mg L−1 ) were nearly the same. The Tmax or time for
Cmax was faster with NanoA (1.58 ± 1.28 h) and NanoB (1.08 ± 0.49 h), compared with
the coconut oil solution (2.33 ± 0.82 h). The bioavailability values of NanoA and NanoB
suspensions following oral administration were 28.6% and 22.7%, respectively, which were
comparable to the coconut oil solution (28.4%), in which CCG-211790 had good solubility.
Pharmaceutics 2021, 13, 1586 12 of 19

Table 6. Pharmacokinetic parameters of the coconut oil solution (n = 6) and NanoA and NanoB
suspensions (n = 6) following oral administration at a dose of 5 mg/kg in male Sprague–Dawley rats.

PK Parameters Coconut Oil NanoA NanoB

Cmax (mg L−1 ) 0.12 ± 0.07 0.13 ± 0.05 0.12 ± 0.03
Tmax (h) 2.33 ± 0.82 1.58 ± 1.28 1.08 ± 0.49 *
AUC0−∞ (mg L−1 h) 0.63 ± 0.27 0.63 ± 0.17 0.50 ± 0.08
MRT0−∞ (h) 6.59 ± 3.22 5.00 ± 1.14 4.34 ± 1.03
t1/2β (h) 3.82 ± 2.23 3.24 ± 1.36 2.61 ± 0.45
Vz /F (L kg−1 ) 46.1 ± 24.6 41.1 ± 21.7 38.2 ± 8.9
CL/F (L h−1 kg−1 ) 8.99 ± 3.14 8.53 ± 2.88 10.14 ± 1.64
FPEG/PG 28.4% 28.6% 22.7%
* p < 0.05, statistically significant difference compared with coconut oil solution.

4. Discussion
In our previous studies, we have identified a series of chemical compounds capable of
inhibiting biofilm formation and virulence factor expression of S. aureus [6,9]. The poor
aqueous solubility of the lead compound CCG-211790 has limited its therapeutic potential.
In the current study, nanotechnology was applied to improve its pharmacokinetic prop-
erties for oral and intravenous delivery, which are common routes of delivery to prevent
and treat wound infections. Two nanosuspension formulations (NanoA and NanoB) of
CCG-211790 were generated using an antisolvent precipitation-ultrasonication method,
a bottom-up approach. NanoA had a smaller particle size and different morphology in
comparison with NanoB. The concentration of Tween 80 is a critical parameter in the
determination of particle size and morphology of CCG-211790 suspensions. CCG-211790
suspensions of the z-average values from 290–350 nm could be reproducibly produced
with the Tween 80 concentration less than 0.02% and the height of water filled in the
ultrasonic tank from 10.4–29.5 mm. CCG-211790 suspensions of the z-average values from
500–1000 nm were obtained with the Tween concentration over 0.02% and the height of
water from 8.1–9.0 mm. The size difference can be explained by the formation of Tween
80 micelles and the change of ultrasonic cavitation intensity with the height of the water.
Critical micelle concentration (CMC) is defined as the concentration above which the dis-
crete monomers of a surfactant start to form micelles, which is 0.0016% (w/v) for Tween 80.
The formation of micelles increases the solubility of hydrophobic drugs by embedding
drug molecules into the hydrophobic cores of the micelles. Therefore, Tween 80 micelles
formed at a high concentration (e.g., 0.02% w/v) would dissolve and reduce the number of
nuclei generated when mixing the organic solution with the antisolvent, which promotes
the growth of particles.
Ultrasonication and mechanical stirring also play important roles for reducing particle
size. Ultrasonication can affect the diffusion coefficients of CCG-211790 molecules via
the creation of acoustic cavitation [53]. Cavitation is a phenomenon in which a large
number of small vacuum bubbles or voids are generated during the high-pressure cycles of
ultrasonic waves and collapse during the low-pressure cycles releasing intense energy [54].
Cavitation is the major driving force for the collision or the fragmentation of existing
crystals [55]. In our previous study, the hydrodynamic particle size (z-average) was reduced
from over 600 nm to less than 400 nm when mechanical stirring from 100–600 rpm was
applied during ultrasonication. Using mechanical stirring simultaneously with sonication,
nanosuspensions of CCG-211790 with two different particle sizes, NanoA (315 ± 6 nm)
and NanoB (915 ± 24 nm) were successfully generated. These nanosuspensions were
demonstrated to be stable for six weeks.
There was a significant increase in dissolution rates for NanoA and NanoB suspensions
compared with CCG-211790 bulk powder. These higher dissolution rates can be explained
by Noyes-Whitney equation: dC DA
dt = hD ·(Cs − C), where dC/dt is the dissolution rate
of a drug from solid state, D is the diffusion coefficient of the drug in bulk solution, A
is the effective surface area of the drug solid in contact with bulk solution, hD is the
Pharmaceutics 2021, 13, 1586 13 of 19

hydrodynamic diffusion layer thickness, Cs is the concentration on the surface of the

dissolving solid and C is the concentration of the drug in bulk solution [56], as displayed
in Figure 7.

Figure 7. Schematic of the diffusion layer proposed by Noyes and Whitney and further modified
by Nernst and Brunner. Particle size reduction leads to increased surface area A and decreased
hydrodynamic layer thickness hD , therefore increasing dissolution rate dC/dt.

Based on the fluid dynamic (FD) theory, the hydrodynamic

  diffusion layer thickness
1/2
hD can be described by Prandtl equation: hD = k· L 1/3 , where L is the length of the
V
surface in the direction of flow (equivalent to the diameter of particles), V is the relative
velocity of the flowing liquid against a flat surface and k is a constant [57].
Though non-FD-based approximations to the hydrodynamic diffusion layer thickness
have also been made by the Hintz-Johnson model and Wang-Flanagan model, it was
mathematically shown that the FD model and non-FD model have similar hD values
for small particles with radii of less than 15 µm [58]. Particle size reduction leads to
increased surface area A and decreased hydrodynamic layer thickness hD , thereby leading
to increased dissolution rate based on Noyes-Whitney equation and Prandtl equation.
Indeed, nanosuspension-based insoluble drugs may have sufficient dissolution rates for
practical intravenous administration [59], and may even behave similarly to their solution
formulations when the particle size is around 100 nm [60].
Although NanoB had larger dimensions in length and width, NanoB showed compa-
rable dissolution rate to that of NanoA at every selected time point. NanoB consisted of
plate- or tube-like particles with the thicknesses of plates or tube walls ranging between
100–300 nm (Figure 2C), which yielded a large surface area, thereby remarkably enhanced
the dissolution rate. In comparison, CCG-211790 bulk powder displayed the slowest
dissolution rate; 19.6% release of CCG-211790 in 5 min, 63.4% in 45 min and 64.3% in
60 min. The low dissolution rate can be explained by the small surface area and the poor
wetting property of the CCG-211790 bulk powder [61]. Unlike CCG-211790 suspensions
that immediately dispersed in the dissolution media, CCG-211790 bulk powder would dis-
perse on the surface for 5–30 min before thoroughly immersing into the dissolution media
when mild stirring (e.g., 75 rpm) was applied. In addition, the high standard deviation
(9.9–20.7%) of CCG-211790 bulk powder dissolution rates at the early time points (from
5–30 min) can also be explained by the random divergence of wetting processes each time.
The pharmacokinetic behavior of drug nanoparticles is deeply associated with their
dissolution properties. Drug nanoparticles that completely dissolve immediately after
Pharmaceutics 2021, 13, 1586 14 of 19

intravenous injection (typically with particles size of sub-200 nm) may have similar phar-
macokinetic profiles to solution formulations [60,62]. In a pilot study of the PK properties
of the powder suspension of CCG-211790, poor bioavailability was observed due to the
poor aqueous solubility of the compound. The pilot study was conducted in feed animals
(food not withheld), while the nanosuspension studies were conducted in fasted animals.
A drug-food effect cannot be predicted on a scientific basis, requiring specific studies to
understand possible effects [63–65]. However, the pilot study did demonstrate that the
bulk compound had poor bioavailability which needed formulation technology to improve
its PK properties.
NanoA and NanoB showed the trend toward lower Cmax 0.8-fold and 0.66-fold re-
spectively, as compared to PEG/PG solution. The lower Cmax and faster clearance of
the nanoparticle formulations may be due to CCG-211790 nanoparticles being slowly
dissolved during the distribution phase. The in vitro dissolution study where complete
dissolution of NanoA and NanoB suspensions occurred within 5 min after exposure to
the dissolution medium indicated a rapid dissolution rate of CCG-211790 nanoparticles
under a sink environment. An additional reason for the change in PK is possibly that
CCG-211790 nanoparticles dissolved rapidly after reticuloendothelial system (RES) uptake
leading to a better volume of distribution and high local tissue concentration. The high
concentration in the liver contributed to the high elimination rate of the compound from
the body via hepatic-metabolism, which may have reduced the concentration to lower
levels. This assumption is supported by the higher clearances (CL) of NanoA and NanoB,
which were 1.8-fold and 2.3-fold respectively to that of PEG/PG solution. This is pos-
sibly due to clearance mechanism of nanoparticles by macrophage/Kupffer cell-uptake
in the liver [66]. The lower plasma concentration at all time points and the lower AUC
(0.58-fold and 0.44-fold, respectively) of NanoA and NanoB suspensions compared to
that of PEG/PG solution could also be explained by this hypothesis. Direct evidence of
nanoparticle uptake in macrophage-rich organs can be determined through histological
analysis, which can be conducted in future work. The Vz s of the nanosuspensions were
higher than PEG/PG solution, suggesting that the compound was well distributed into the
body, and as an anti-virulence agent could act against the bacteria within the host tissues
and organs for wound infection clearance and prevention of further complications such as
systemic infections.
The oral PK properties of the nanosuspensions were also studied to assess their poten-
tial as oral formulations because oral delivery is the most preferred route of drug delivery.
The epithelia cells (e.g., intestinal epithelium) in the gastrointestinal (GI) tract function as a
biological barrier that allow small molecules to pass through but block macromolecules or
particles from entering systemic circulation [67]. Bioavailability of a water-insoluble drug
can be enhanced by sufficient release of drug molecule from solid states or solubilizing
vehicles into body fluids [68]. According to the Noyes-Whitney equation, the dissolution
rate of drug nanoparticles can be significantly increased due to the large surface area,
increasing GI uptake [69]. Furthermore, drug nanoparticles’ adhesion to biological mucosa
in the GI tract can lead to accumulation on the GI mucosal surface enhancing retention for
enhanced absorption [70]. While the Cmax s for all formulations were nearly the same as
shown in Table 6 and Figure 6, the Tmax s or time for Cmax s were faster with the nanoparticle
formulations and indicated that the drug would get into the blood and be distributed into
the tissues faster, suggesting the nanosuspensions would have potentially faster onset of
activity against the bacteria than solution formulations. Encouragingly, the bioavailability
of NanoA or NanoB suspensions following oral administration was comparable with that
of the coconut oil solution. Thus, converting water insoluble CCG-211790 into nanosuspen-
sions could improve the PK properties of the compound to the levels of that of an oil-based
solution.
In this study, PEG/PG solution was designed for intravenous administration. How-
ever, PEG/PG solution has no clinical potential because of limited solubility of powder
CCG-211790 (up to 1 mg/mL) and the solution had poor storage stability with precipitation
Pharmaceutics 2021, 13, 1586 15 of 19

occurring within one day. Coconut oil can be used as the vehicle for oral formulations with
the solubility of CCG-211790 up to 37 mg/mL [9] but is not suitable for intravenous admin-
istration. CCG-211790 nanosuspensions, particularly NanoA, have exhibited good storage
stability, enhanced oral bioavailability comparable with coconut oil solution, good tissue
distribution and the potential for both oral and intravenous administrations. Therefore,
improved Cmax , tissue distribution and overall bioavailability for an anti-virulence agent
could contribute to a clinical benefit for controlling bacteria without the need for oils or
other excipients that may cause some tolerability/safety problems for oral administration
and IV use.
Additionally, a drug candidate with poor aqueous solubility possesses low and highly
variable oral bioavailability. As a result of the low oral bioavailability, the drug candidate
must be administrated in a larger dose than required if it is to have higher bioavailabil-
ity to achieve a therapeutically active concentration. Improving its bioavailability via
nanosuspension approach could reduce the administered dose, thus decreasing the poten-
tial side-effects due to excessive drug dumping in the body, resulting in a lower cost of
therapy [71–73].
The nanosuspensions can be formulated in diverse dosage forms to achieve higher
loading capacities via different formulation methods such as wet milling, high-pressure
homogenization, and nanocrystal powder-loaded tablets, which have the potential to
further improve their bioavailability and exposure in tissues [33,34]. In addition, the
pharmacokinetic properties of CCG-211790 nanosuspensions can be further optimized by
surface modification with, for example, layer-by-layer assembled polyelectrolytes [74] or
by coating with enteric polymers for controlled and extended release [75].

5. Conclusions
CCG-211790 nanosuspensions with different particle sizes and morphology, termed as
NanoA (z-average of 315 ± 6 nm) and NanoB (z-average of 915 ± 24 nm), were produced
using antisolvent precipitation-ultrasonication method. The majority of particles in NanoA
were small particles with dimensions from 200–400 nm, while the dominant particles
in NanoB were plate- or tube-like particles with lengths up to a few micrometers but
thicknesses down to a hundred nanometers. Both NanoA and NanoB nanoparticles carried
high magnitudes of negative zeta potential (up to 40–60 mV) and could maintain physical
stability over 6 weeks when stored at 4 ◦ C or room temperature. NanoA and NanoB
suspensions exhibited markedly enhanced in vitro dissolution rates, with almost 100%
release within 5 min and particle-like behaviors (e.g., lower Cmax s and larger Vz s) during
the distribution phase following intravenous administration in rats. NanoA and NanoB
suspensions had oral bioavailability values of 28.6% and 22.7%, respectively. Bioavailability
was comparable with that of the solution in a coconut oil formulation. Therefore, NanoA
suspension is a promising formulation for the oral and intravenous administration for
poorly soluble drugs, particularly for oral drug delivery, which is the most preferred
administration route because it is noninvasive and can be self-administrated with high
patient compliance, making it suitable for repeated and prolonged treatment outside a
hospital setting. The pharmacokinetic properties can be further improved by using various
approaches to enhance its potential therapeutic applications [76].
We have demonstrated the application of nanoparticle technology to improve the
dissolution rate and overall bioavailability of a very insoluble antibacterial compound,
CCG-211790 for treating wound infections. Using this approach in the future for drug can-
didates, it may be possible to generate products requiring lower doses, with reduced food
effects, more rapid onset-of-action, and many other beneficial outcomes. Nanoparticles can
target antimicrobial agents into tissues more efficiently. This can improve drug concentra-
tion at the site of infection, so that higher doses of the drug are achieved at the infected
site, thereby potentially overcoming resistance. Additional nanoparticle-based strategies
are providing designs for enhanced delivery of drugs for important therapies, including
antibiotic-resistant bacteria in chronic wound infections and cancer [77–80]. Many of these
Pharmaceutics 2021, 13, 1586 16 of 19

formulation technologies are targeted delivery systems, directing the therapeutic agent
directly to the target cells-bacteria/bacteria infected cells or cancer cells. The future of more
effective and safer treatments for many diseases will be impacted by nanotechnology.

Author Contributions: Conceptualization, D.W.A., H.L. and H.S. (Hongmin Sun); methodology,
N.W., F.Q., X.H., H.S. (Honglan Shi), D.W.A., H.L. and H.S. (Hongmin Sun); formal analysis, N.W.,
F.Q., X.H., H.S. (Honglan Shi), D.W.A., H.L. and H.S. (Hongmin Sun); investigation, N.W., F.Q.,
X.H. and H.S. (Honglan Shi); writing—original draft preparation, N.W., F.Q., X.H., H.S. (Honglan
Shi), D.W.A., H.L. and H.S. (Hongmin Sun); funding acquisition, H.L. and H.S. (Hongmin Sun). All
authors have read and agreed to the published version of the manuscript.
Funding: This work was supported by Nanova Inc. in the form of a gift fund for the Curators of
University of Missouri awarded to H.L. and H.S. (Hongmin Sun), and expenses for performing PK
study of CCG-211790 suspension formulation at Gateway Pharmacology Laboratories and Ivogen
Inc. in the form of a salary for D.W.A. The funders had no role in study design, data collection and
analysis, decision to publish, or preparation of the manuscript.
Institutional Review Board Statement: The animal experimental procedures (protocol 9650) were
approved by the Animal Care and Use Committee (ACUC) of University of Missouri (Columbia,
MO, USA).
Informed Consent Statement: Not applicable.
Data Availability Statement: Data are contained within the article.
Acknowledgments: We would like to thank Haiqing Yu, Lisa Watkinson, and Terry Carmack for
assisting in the animal PK studies and John E. Jones for helping with proofreading the manuscript.
Conflicts of Interest: D.W.A. and H.L. are currently employed by Ivogen Inc., which is a subsidiary
of Nanova, Inc. H.S. (Hongmin Sun) is a consultant of Nanova Inc., Ivogen Inc., and owns stocks in
Nanova, Inc. F.Q. is currently employed by BeiGene Ltd. X.H. is currently employed by Frontage
Laboratories, Inc. The companies had no role in the design of the study; in the collection, analyses,
or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.
The authors would like to declare the following patents associated with this research for methods
and compositions for treating bacterial infection: H.S. (Hongmin Sun) is a co-inventor on patents
US 8501722, US 9504688, Japan 6293736, and European 2844258; H.S. (Hongmin Sun) and DWA are
co-inventors on patent US 9814719; H.S. (Hongmin Sun), D.W.A., and F.Q. are co-inventors on patent
US 10441588. Ivogen Inc. is developing products related to these patents. This does not alter the
authors’ adherence to all the journal’s policies on sharing data and materials.

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