Cyclodextrin-erythromycin complexes as a drug delivery device for orthopedic application

Figures & data

Figure 1 500 mHz ¹H-NMR spectra at 25°C of (A) cyclodextrin 14 mg (B) erythromycin 6 mg (C) cyclodextrin-erythromycin 20 mg (MV) (7/3, w/w) complex, (D) 20 mg of cyclodextrin-erythromycin (MVI) (7/3, w/w) complex in 750 μL of D₂O ranging from 0.5 to 5.5 ppm. Important proton chemical shifts and peak integrations of solutes are labeled.

Abbreviations: MV, method V; MVI, method VI.

Figure 2 500 mHz ¹³C-NMR spectra at 37°C of 30 mg of cyclodextrin, 30 mg of cyclodextrin-erythromycin (MV) (7/3, w/w) complex, 30 mg of cyclodextrinerythromycin (MVI) (7/3, w/w) complex in 1 mL of D₂O. Important carbons (C1–C6 and C from erythromycin) chemical shifts and peak integrations of solutes are labeled.

Abbreviations: CD, cyclodextrin; CD-EM, cyclodextrin-erythromycin; MV, method V; MVI, method VI.

Table 1A Proton chemical shift of cyclodextrin

Proton	H1	H2	H3	H4	H5	H6
Free-CD	4.953	3.543	3.852	3.471	3.744	3.764
CD-EM (MV)	4.951	3.540	3.847	3.467	3.736	3.761
CD-EM (MVI)	4.951	3.540	3.848	3.467	3.736	3.761
δ(MV)	−0.002	−0.003	−0.005	−0.004	−0.008	−0.003
δ(MVI)	−0.002	−0.003	−0.004	−0.004	−0.008	−0.003

Abbreviations: CD, cyclodextrin; CD-EM, cyclodextrin-erythromycin; MV, method V; MVI, method VI.

Table 1B Proton chemical shift of erythromycin

Proton	3′	4	6	7′	8	10	12	19	21
Free EM	0.722	2.016	1.486	1.814	2.296	3.191	3.104	2.967	1.122
MV	0.734	2.014	1.488	1.785	2.243	3.204	3.110	2.961	1.060
MVI	0.736	2.014	1.492	1.783	2.235	3.204	3.112	2.968	1.060
δ(MV)	+0.012	−0.002	+0.002	−0.029	−0.053	+0.013	+0.006	−0.005	−0.062
δ(MVI)	+0.014	−0.002	+0.006	−0.031	−0.061	+0.013	+0.008	+0.001	−0.062

Note: Chemical shift variations with significance were specified in bold type.

Abbreviations: EM, erythromycin; MV, method V; MVI, method VI.

Table 1C Carbon chemical shift of cyclodextrin

Carbon	C1	C2	C3	C4	C5	C6
Free-CD	101.884	81.202	73.150	72.143	71.906	60.394
CD-EM (MV)	101.892	81.202	73.150	72.143	71.906	60.387
CD-EM (MVI)	101.892	81.202	73.150	72.143	71.906	60.387
δ(MV)	0.008	0	0	0	0	0.007
δ(MVI)	0.008	0	0	0	0	0.007

Note: Chemical shift variations with significance were specified in bold type.

Abbreviations: EM, erythromycin; CD, cyclodextrin ; MV, method V; MVI, method VI.

Figure 3 Three-dimensional molecular structure of cyclodextrin-erythromycin complex. Yellow, erythromycin; pink, cyclodextrin 1; green, cyclodextrin 2; blue, cyclodextrin 3.

Figure 4 X-ray diffraction spectra for pure cyclodextrin, cyclodextrin-erythromycin (MV and MVI) before freeze-drying, and pure erythromycin. The Y axis (counts) of the diffraction pattern is vertically offset for clarity. Blue-peaks disappeared in spectra of CD-EM complex. Red-new peaks in spectra of CD-EM complex. Yellow-characteristic peaks of EM in in spectra of CD-EM complex.

Abbreviations: CD, cyclodextrin; EM, erythromycin; MV, method V; MVI, method VI.

Figure 5 Ultraviolet spectra of (A) pure erythromycin, (B) pure cyclodextrin, (C) cyclodextrin-erythromycin (MV), and (D) cyclodextrin-erythromycin (MVI) from 200 nm to 300 nm with a step size of 1 nm.

Abbreviations: MV, method V; MVI, method VI.

Figure 6 Scanning electron microscopic morphologies of (A, 100×; D, 300×) cyclodextrin, (B, 300×; E, 1000×) erythromycin and (C, 300×; F, 1000×) cyclodextrinerythromycin (MVI).

Abbreviation: MVI, method VI.

Figure 7 Drug stability profiles of (A) erythromycin and (B) cyclodextrinerythromycin dissolved in 5 mL D-H₂O at 37°C.

Note: Measurable percentage of erythromycin against time; n = 3.

Abbreviations: CD, cyclodextrin; EM, erythromycin.

Figure 8 Bactericidal effect assay. (A) Semiquantitative bacteria growth inhibition assay in 2 mL broth in the presence of cyclodextrin-erythromycin with gradient concentrations (53, 106, 159, and 212 μg/mL) for 24 hours, respectively. (B) Semiquantitative bacteria growth inhibition assay in 2 mL broth in the presence of erythromycin 64 μg/mL, cyclodextrin 148 μg/mL, and cyclodextrin-EM 212 μg/mL at predetermined time points, respectively.

Notes: All dots were exponentially fitted; P < 0.05 related to 0 versus 159 and 212 μg/mL; n = 4.

Abbreviations: CD, cyclodextrin; EM, erythromycin.

Figure 9 MC-3T3 cell activity assay. The live (green)-dead (red) staining (original magnification, ×50) of MC3T3 cells cultured for 16 days in the presence of (2) erythromycin 1.6 mg/mL, (3) cyclodextrin 3.7 mg/mL, and (4) cyclodextrin-erythromycin 5.3 mg/mL. ALP activity of MC-3T3 cells cultured under the same conditions in the presence of drugs with (A) higher concentrations of erythromycin (1.6 mg/mL), cyclodextrin (3.7 mg/mL), and cyclodextrin-erythromycin (5.3 mg/mL) and (B) lower concentrations of erythromycin (16 μg/mL), cyclodextrin (37 μg/mL), and cyclodextrin-erythromycin (53 μg/mL).

Note: *P < 0.05 related to control versus cyclodextrin and cyclodextrin-erythromycin; n = 6.

Abbreviations: ALP, alkaline phosphatase; CD, cyclodextrin; EM, erythromycin.

Figure 10 Erythromycin inhibits the amount of RANKL-induced TRAP⁺ cell formation in murine RAW 264.7 cells. (A–E) Representative TRAP staining (original magnification, ×100): (A) RANKL (−); (B) RANKL (+); (C) RANKL (+) + erythromycin (16 μg/mL); (D) RANKL (+) + cyclodextrin (37 μg/mL); and (E) RANKL (+) + cyclodextrin-erythromycin (53 μg/mL). TRAP⁺ cells were quantified by Image Analysis software (bottom panel). The value represents TRAP⁺ cells location quantified as area percentage of TRAP⁺ cells (%). Values represent averages of triplicate cultures from one representative experiment.

Notes: Using analysis of variance, *P < 0.05 refers to RANKL (+) + cyclodextrin 37 μg/mL versus RANKL (−), RANKL (+) + erythromycin 16 μg/mL, and RANKL (+) + cyclodextrin-erythromycin 53 μg/mL; **P < 0.05 related to RANKL (+) + cyclodextrin-erythromycin 53 μg/mL versus RANKL (−); n = 3.

Abbreviations: CD, cyclodextrin; EM, erythromycin; RANKL, receptor activator of nuclear factor kappa-B ligand.

Figure 11 Lactate dehydrogenase activity in (A) culture medium of MC-3T3 cells after drug treatment [higher concentrations of erythromycin (1.6 mg/mL), cyclodextrin (3.7 mg/mL), and cyclodextrin-erythromycin (5.3 mg/mL)] for 6, 11, and 16 days, n = 6; (B) in culture medium of RAW 264.7 cells after drug treatment [lower concentrations of erythromycin (1.6 ug/mL), cyclodextrin (3.7 ug/mL), and cyclodextrin-erythromycin (5.3 ug/mL)] for 4 and 7 days, n = 3.

Note: The values shown are the mean ± standard error of the mean from six observations.

Abbreviations: CD, cyclodextrin; EM, erythromycin; LDH, lactate dehydrogenase.