Biomaterials Volume 18 issue 4 1997 [doi 10.1016%2Fs0142-9612%2896%2900144-5] V. Masson; F. Maurin;...

8/10/2019 Biomaterials Volume 18 issue 4 1997 [doi 10.1016%2Fs0142-9612%2896%2900144-5] V. Masson; F. Maurin; H. F

1/9

PII SO142-9612 (96) 00144-S

Biomoteriol.5 18 (1997) 327-335

0 1997 Elsevier Science Limited

Printed in Great Britain. All rights reserved

014%9612/97/ 17.00

Influence of sterilization processes on

poly( -caprolactone) nanospheres

V. Masson, F. Maurin, H. Fessi and J.P. Devissaguet+

Laboratoire Chauvin S.A., BP 1174,34009 Montpellier cedex 1, France; Labora?oire de GalBnique, ISPB, llniversite

Lyon 1.8 Avenue RockefeUer, 69008 Lyon, France; Laboratoire de Pharmacotechnie et Biopharmacie, URA CNRS

1218, Universit Paris XI, 92290 Chstenay-Malabry, France

Polymeric vectors and especially poly(e-caprolactone) nanoparticles have already shown promising

results in the optimization of the ophthalmic bioavailability of drugs. Any formulation instilled in the

eye must be sterile, and preferentially isotonic. Poly(&-caprolactone) nanospheres were thus

formulated with Synperonic PE/F68, Synperonic PE/F127, or Cremophor RH40. A tonicity agent, a

preservative and, in some cases, a viscosifiant were then added. The pH was finally adjusted to pH 4

or buffered to pH 7. Different sterilization processes were studied to investigate their influence on the

physicochemical characteristics of vectors. Autoclaving did not induce any modification on polymer

molecular weight or Synperonic nanospheres diameter, but catalysed some reactions with surfactants

and tonicity agents. This method could thus be used if the nanosphere excipients are chosen with

care. J radiation induced preservative degradation and viscosifiant depolymerization. A cross-linking

of poly(e-caprolactone) chains was observed, as reflected by a sharp increase of its molecular weight.

However, no variation of the mean particle size was detected. Finally, sterile filtration was the only

process which ensured the conservation of physicochemical integrity of nanospheres. This process

was successfully applied on non-viscosified vectors with a sufficiently small diameter. 0 1997 Elsevier

Science Limited. All rights reserved

Keywords: Poly (c-caprolactone), nanospheres, moist sterilization, y irradiation, sterile filtration

Received 28 February 1996; accepted 8 August 1996

Application of drugs to the eye is usually achieved by

instillation of conventional eye drops. This generally

results in extensive drug loss owing to tear turnover,

lacrymal drainage, blinking and drug dilution by

tears.

In the past decade, many authors have studied

several systems to increase the precorneal residence

time of drugs, and prolong their penetration into the

intraocular structures. Insertsz4, hydrogels5-7,

bioadhesive polymers5r6Z8, liposomesg-I, microemul-

sions12*13 and nanoparticles14-*6 have therefore been

proposed. Among these, polymer nanospheres and

nanocapsules have shown their capacity to enhance

the ocular therapeutic effect of associated drugs15Z7-21.

Any formulation instilled in the eye must be sterile.

Production

of polymeric vectors under aseptic

conditions is possible but complex and expensive. All

materials are sterilized by dry heat before use and a

sterile filtration of the polymerization medium is

carried out22-24. In any case, terminal sterilization

would be preferred, since aseptic processing is more

risky with respect to microbial contamination of the

finished product.

To our knowledge,

sterilization of submicronic

particles by dry heat has never been reported.

Gelatinz526, polymethacrylicz7

and polycyanoacrylicz8

Correspondence to Dr V. Masson.

nanoparticles have been moist sterilized without

variation of the mean size of the vector. Nevertheless,

Rollot et

a l

observed an important increase of the

diameter (from 200 to 500nm) after heat sterilization

of nanocapsules, prepared with miglyol surrounded by

poly(isobutylcyanoacrylate). The size modification

could have resulted either from a swelling of the

polymeric membrane or from an expansion of the oily

phasezg.

Gamma irradiation can also affect the performance of

a drug delivery system (DDS)30. Firstly, radiolytic

degradation of the drug substance may cause the

formation of potentially toxic by-products, thereby

reducing the nominal drug content. Secondly,

degradation of the polymer may have consequences

both for drug release from the DDS and resorption of

the device under

in vivo

conditions. Moreover, the

shelf-life and stability can be reduced by this step.

However, this process has been applied to the steriliza-

tion of polymeric microspheres30-33.

Alleman et al. have also studied the influence of

y irradiation on

the properties of polymeric

nanospheres. Polylactic nanoparticles loaded with

CGP 19486 or with savoxepine were sterilized by a

2.5 Mrad dose of y radiation. The mean size of the

vector was not altered but a significant increase in

drug release rate was observed. This was apparently

due to a dramatic decrease in the number average

327

Biomaterials 1997, Vol. 18 No. 4


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328

Sterilization of poly(E-caprolactone) nanospheres:

V. Masson et a

molecular weight of the polymer, after sterilization

doses above 1 Mrad4,5.

To avoid any structural or physicochemical modifica-

tions during sterilization, the best process is sterile

filtration on

0.22

pm filters. Several authors stated that

this method cannot be used, since the nanoparticles

are similar in size to contaminants and pore size of

filters. Moreover, the elasticity of the particles could

lead to the clogging of the filtration membranes34z3fi.

No motivation was thus provided for testing this

sterilization procedure.

The aim of the present work was to study the

influence of sterilization

processes on the

physicochemical characteristics of poly(s-caprolactone)

(PCL) nanospheres. This polymer was used with

success for the optimization of the administration of

carteolol, betaxolol and indomethacin in the eye. To

select a proper sterilization method, we studied the

influence of autoclaving, y irradiation and sterile

filtration on different formulations of poly (s-caprolac-

tone) nanospheres.

This organic solution is then poured in an aqueous

phase,

containing the hydrophilic surfactant at a

concentration of 0.4%. The aqueous phase immediately

turns milky, with bluish opalescence as a result of the

formation of nanospheres. The acetone and a part of

the water is finally removed by evaporation under

reduced pressure to obtain a concentration of 1.56% of

polymer and 2.5% of surfactant. One batch, for each

polymer/surfactant association, was prepared. Each

batch was divided into four aliquots. Glucose,

thimerosal, HEC, phosphate buffer or HCl were then

added as concentrated solutions. Finally the volume of

the preparation was adjusted, to obtain a polymer

concentration of 1.25%.

Sterilization

M TERI LS ND METHODS

Materials

Poly(c-caprolactone) with molecular weights (Mw) of

60000 (PEC) and 150000 (Tone P787) were purchased

respectively from Aldrich Chimie SARL (Strasbourg,

France) and Union Carbide France SA (Rungis,

France).

Polyoxyethylene-polyoxypropylene block

copolymers, synperonic PE/F68 (PF68) and PE/F127

(PF127), were provided by ICI (Verfilco, Fontenay sous

Bois, France), and Polyoxyethylene (40) hydrogenated

castor oil (Cremophor RH40) (CRH40) by BASF

Corporation (Levallois-Perret, France). Hydroxyethyl-

cellulose (Natrosol 250XH Pharm) (HEC) was obtained

from Aqualon. Thimerosal was purchased from Sigma

Chimie (St Quentin Fallavier, France), and glucose

monohydrate from Prolabo. All other reagents were of

analytical grade.

Autoclaving and y ir radiation. 40ml of each

formulation was prepared and placed in

20

glass vials,

which were then sealed with rubber stoppers and

aluminium caps. Half of the samples were sterilized at

121C for 20min (BMA liquid S93 sterilizer, Breux

Board & Cie, Montpellier, France), or by y irradiation

(2.5MRad) using a

fioCo source (Ionisos, Dagneux,

France). The other half of the samples were maintained

at 4C until being controlled. These controls were

carried out at the same time on both sterilized and

non-sterilized batches, in order to take the aging of

preparations into account.

Sterile filtration.

Since the diameter of PEC

nanospheres was below 200nm, a sterile filtration on

0.2 pm filter was tested. In the case of Tone P787

formulations, the mean size was too high (=220nm)

to allow this process. The high viscosity of

preparation containing HEC seems to induce a

clogging of membranes. In fact, even after a prefiltra-

tion on 2pm, 1 pm and 0.8pm, only a 0.45pm filter

could be used.

Preparation of nanospheres

Formulations

Various formulations of nanospheres were prepared

using different kinds of polymers and surfactants.

Nanospheres of PEC were manufactured with PF68,

PF127 or CRH40 as surface active agents. In the case of

Tone P787, only CRH40 was used as this surfactant

gave rise to the smallest nanospheres diameter. Each

formulation (association polymer/surfactant) was

adjusted to pH 4 or buffered to pH 7, in order to

evaluate the role of the original pH on the stability of

nanospheres, during the sterilization process.

0.3%

of

HEC was added in some batches in order to prevent a

possible sedimentation

of particles. Thimerosal

(0.01%) was added as antimicrobial agent, except in

pH 4 preparations, as this product may precipitate in

acidic conditions7.

Finally, isotonicity was obtained

after the addition of glucose in adequate quantities.

Finally,

the following process was retained.

Prefiltration: glass fibre filter, 2 pm (Ultipor GF Plus

U22OZ, PALL, St Germain en Laye, France), and

cellulose acetate filter, 0.45 ,um (Minisart NML,

Sartorius, Palaiseau, France). Filtration: cellulose

acetate filter, 0.20 pm (Minisart NML, Sartorius,

Palaiseau, France).

This method was tested only on PEC nanospheres,

without HEC, adjusted to pH 4.

Nanospheres evaluation

The influence of autoclaving, irradiation and sterile

filtration on nanosphere characteristics was evaluated

by the study of visual appearance and particle sizes of

the vectors. In the case of heat sterilization and y

radiation, pH, tonicity and molecular weight of poly(e-

caprolactone) were additionally analysed, as these

processes may affect the properties of the different

constituents, especially the polymer. In the case of

sterile filtration, a study of nanosphere turbidimetry

was undertaken in order to estimate the concentration

of particles, before and after filtration.

Preparation

Visual appearance

Nanospheres were prepared according to the method

developed by Fessi et a1.3833g.Briefly, the polymer is

The nanosphere dispersions were characterized before

and after sterilization with respect to sedimentation,

dissolved in acetone to a final concentration of

0.5%.

flocculation, aggregation and colour.

Biomaterials 1997,Vol. 18 No. 4


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Sterilization of poly(E-caprolactone) nanospheres: V Ma sson et a/.

329

Particle size

RESULTS ND DISCUSSION

The mean particle size and size distribution were

determined by photon correlation spectroscopy using a

nanosizer (N4MD, Coultronics, France).

utoclaving

PH

pH of the suspensions was measured with a Mettler

delta

320

pH meter.

Tonicity

Tonicity

was

evaluated by a freezing-point

measurement using a Fiske MS TM cryoscope

(Radiometer Tacussel SA, Neuilly-Plaisane, France).

No measure was carried out on samples containing

HEC, as their viscosity was too high to allow any

analysis.

Heat sterilization, like y irradiation, can affect the

physicochemical properties of nanosphere constitu-

ents, thus influencing the stability of the particle itself.

In fact, as shown in

Table

1, sterilization of cremophor

RH40 formulations induced a massive aggregation of

particles,

whatever the

polymer

used.

This

phenomenon could result from the lower cloud point

of this surfactant (95.6X), compared with PF68 or

PF127 (>looC).

Poly mer molecular w eight

The molecular weight of poly(E-caprolactone) inside

nanospheres was determined by gel permeation

chromatography, using a Waters Associates chromato-

graphy system fitted with two Ultrastyragel columns,

104w and

5OOk

Tetrahydrofuran (THF) was used as

the eluent, with a flow rate of 1 ml min-. A differential

refractometer (Waters 410) was used (sensitivity= 16,

temperature= 35C) and the elution profile was

acquired through interfacing with a Data Module

Waters 730. Column temperatures were maintained at

40C with a Waters TM Temperature Control-System.

A calibration was carried out using a series of six

polystyrene standards in the molecular range

1800-

354 000. Experiments were performed on sterilized

and non-sterilized preparations after freeze drying, in

order to remove any aqueous phase. The lyophilizates

were resuspended in THF (final concen-

tration = 0.625% W/V of polymer), and filtered through

a

0.45ltm

filter (Minisart SRP4, Sartorius). A 100/d

aliquot was then injected for analysis. A shoulder

appeared between polymer and surfactant peak. The

polymer peak was thus integrated using the valley as

the last slice. In any case, no Mn value was calculated,

as the number average molecular weight is greatly

affected by low molecular weights.

Indeed, the aggregation upon heating is directly

related to the precipitation and/or phase separation of

the surface modifier at a temperature above its cloud

point, where this molecule is likely to dissociate from

the particle. The unprotected vector can then aggregate

in clusters. Upon cooling, the surfactant redissolves in

the solution and coats the aggregated particles,

preventing them from dissociating into smaller

ones41-44. Several authors have thus recently proposed

the use of different substances to increase the cloud

point of surfactant, above the temperature required for

sterilization (for example PEG 400, propylene glycol,

phospholipid)4144.

Synperonic nanospheres did not show any visual

modification except a yellowish colouration which

appeared in buffered samples. These coloured samples

also showed an increase of tonicity and an acidifica-

tion, In fact, the pH of the different buffered

preparations decreased from 7.15 to about 6.30

(Figure

z), and their delta value changed from -0.635 to

-0.665. The heat treatment of preparations containing

glucose, in the presence of weak bases such as salts of

organic acids, leads to the degradation of the polyol

with oxidation and isomerization.

Furthermore, an increase in basicity of the salt

induces an increase in the level of isomerization. All

these phenomena involve the formation of brown

products and an acidification of the medium4546.

Turbidimetric measurements

Turbidimetry of nanosphere

suspensions

was

evaluated, before and after filtration, to estimate a

possible retention of vectors on filter membranes4.

The transmission percentage was measured at 400nm,

on a PU 8720 UV/VIS scanning spectrophotometer

(Philips), after a 1:80 dilution of the sample.

Disodium phosphate used to buffer the preparation to

pH 7 may have reacted with glucose according to this

process.

In order to confirm this hypothesis, we

prepared different blends of nanosphere constituents,

and sterilized them by autoclaving. The formulations

were tested, and results obtained are summarized in

Table 2.

These experiments confirmed well the interaction

between glucose and disodium phosphate, thus

leading to a yellowish colouration, an acidification and

an increase of tonicity, as for buffered nanoparticles.

Table 1 Nanospheres aspect evolution after autoclaving

Polymer:

Surfactant:

PEC

PF68

PF127

CRH40

Tone P787

CRH40

pH4

pH4 + HEC

-

-

-

-

Aggregation

Aggregation

Decrease in viscosity

pH7 buffer

pH7 buffer + HEC

Yellowish

colouration

Yellowish

colouration

Yellowish

colouration

Yellowish

colouration

Aggregation

Yellowish colouration

Aggregation



Aggregation

Aggregation


Aggregation


Aggregation



Biomaterials 1997,

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Sterilization of poly(e-caprolactone) nanospheres: V. Masson et al .

Table 2

Evolution of physicochemical characteristics of different blends of nanosphere constituents after autoclaving

Formulations Visual aspect

PH

n (C)

evolution

Before

After

Before After

autoclaving

autoclaving autoclaving

autoclaving

PF127

-

6.11 4.67

-0.015

-0.009

Glucose

-

6.35 4.60

-0.335

-0.327

Thimerosal

-

6.31 6.31

-0.006

-0.000

pH 7 buffer

-

7.16 7.16

-0.161

-0.164

PF127 +

-

7.17 7.17

-0.186

-0.185

pH 7 buffer

PF127 +

-

5.93 3.87

-0.358

-0.363

Glucose

Glucose +

yellowish

7.12 6.61

-0.506

-0.524

pH 7 buffer

colouration

Glucose +

-

6.33 4.53

-0.331

-0.326

thimerosal

Thimerosal+

-

7.15 7.15

-0.163

-0.165

pH 7 buffer

Glucose + -

4.95 4.49

ND

ND

NaH,PO,

Glucose +

yellowish

9.10 6.28

ND

ND

Na2HP04

colouration

PF127 +

yellowish

7.14 6.65

-0.550

-0.567

Glucose +

colouration

pH 7 buffer

PF127 +

-

7.17 7.15

-0.187

-0.189

Thimerosal+

pH 7 buffer

Glucose +

yellowish

7.12 6.60

-0.508

-0.529

Thimerosal+

colouration

pH 7 buffer

Concentrations of the different constituents: PF127: 2%, glucose.1 H20: 4.03%, thimerosal- O.Ol%, pH 7 buffer = NaHzPOd 2 HzO: 0.17% t Na~HP04.12 HzO. 0 83%.

(n = 3) (ND = not determined).

This hypertonicity was observed only in buffered

vectors, and in blends containing glucose and pH 7

buffer. This may be due to the apparition of ionized

oxidized products of glucose in the medium. This

reaction could also explain the greater decrease of pH

observed in sterilized buffered samples

=0.8),

compared to acidic ones

~0.5) (see Figure 1).

No sodium salt was introduced in pH 4 samples, as

the pH was adjusted with HCl. Thus, the pH decrease

observed could not result from a degradation of

glucose. Other ingredients like surfactants or polymer

may have produced acidic substances during steriliza-

tion.

Siedenbrodt et al. have studied the pH stability of a

10 synperonic solution in water, or in Sorensen pH

7.4 phosphate buffer, after heat treatment. They

observed a decrease in pH, even in buffered solutions47.

In fact, like most non-ionic surfactants, synperonics

t -0,2

8

:,

;

-O/f

%

-0,6

-0,6

-l,o

pai4

pH4 HEC

pH7 buffer

pH7 buffer HEC

Figure 1 pH evolution in autoclaved preparat ions (B PEC/PF68,0 PEC/PF127, 0 PEUCRH40, one P787KRH40).

Biomaterials

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Vol. 18 No. 4


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Sterilization of poly(&-caprolactone) nanospheres: V. Massan et a/.

331

may gradually degrade

if placed in oxidizing

conditions. The initial products of oxidation are

believed to be aldehydes, which if allowed to degrade

completely can be transformed to carbon dioxide and

water48.

An oxidation of polyoxyethylene units has

also been reported for Cremophor R&IO and may be

responsible for an acidification of the medium.

Additionally, poly(&-caprolactone) itself could have

been hydrolysed by heat treatment with subsequent

apparition of free carboxylic end groups. In any case,

this phenomenon is unlikely as Mw of the polymer

remained

constant during sterilization of

all

formulations. It seems that the polymer has conserved its

integrity and therefore its degradation could not explain

the aggregation of cremophor

RH40

nanospheres. The

upper melting point of poly(s-caprolactone) is about

58C, and above this temperature the mechanical

properties of the polymer decline. PCL is thermally

stable, although above 220C it slowly depolymerizes to

yield caprolactone monomer and a crystalline dimer4.

Ouhadi

et al.

have studied the influence of heat

treatment at 120C in air, on the chemical stability of

poly(s-caprolactone). These authors observed that the

intrinsic viscosity of the polymer decreases

significantly after a short period of time (~1 day). The

thermal treatment seemed to induce two simultaneous

processes: a radical initiated thermo-oxidative

degradation and a statistical ester interchange reaction.

The induction period probably corresponds to the

period of time required for the transformation of the

OH end groups into hydroperoxide functions5. The

period of 20min used for autoclaving thus seems too

short to allow this process.

The mean particle size (MPS) of PEC nanospheres

was about 130nm with Cremophor RH40 and 160-

180nm with Synperonics. In the case of Tone P787,

MPS increased to about 230nm. The nanosphere

diameter was measured before and after autoclaving,

except in formulations containing CRH40, where an

aggregation occurred. In all other suspensions, no

variation of particle size was observed.

y irradiation

The main variation of suspension visual aspect, after

irradiation, was the apparition of a dark sediment in

pH 7 batches, and the loss of viscosity for the

formulations containing HEC.

The dark precipitate is probably related to the

degradation of thimerosal with formation of inorganic

mercury salts. Blackburn et al. have shown that both

phenylmercuric acetate and chloride undergo consider-

able degradation after y irradiation of dilute neutral

solutions51. As there was no thimerosal in formulations

adjusted to acidic pH, no precipitate was observed in

these preparations.

All nanospheres containing HEC lost their viscosity

upon y radiation treatment. The same phenomenon

was observed by Sebert et al. after y sterilization of

different aqueous solutions of hydroxypropylmethyl-

cellulose. The decrease in viscosity was well correlated

to the y radiation dose. Irradiations seem to have a

stronger effect on secondary structure (conformation of

polymerized molecule) than on primary structure

(polymer functions and chemical groups). The main

effect was in fact depolymerization5.

The suspensions tonicity remained constant upon

irradiation.

The evolution of poly(s-caprolactone) molecular

weight in nanospheres after y radiation is presented in

Figure 2 All the formulations showed an increase of

Mw, especially in nanospheres prepared with the high

molecular weight polymer (Tone P7870). Narkis has

studied the structure and physical properties of y

irradiated poly(caprolactone) as a function of the

radiation dose leve153. GPC results showed that

irradiation of PCL, like for other semicrystalline

polymers, is a selective process. The radiation induced

reactions occur preferentially in the amorphous

regions,

causing scission (1 Mn) and cross-linking

(~Mw) of chains. In poly(caprolactone), the ratio of

scission to cross-linking events was found to be about

unity54-5.

The increase of Mw observed here could be

a result of the cross-linking between polymeric chains

or even between surfactant and poly(caprolactone)

chains.

Tone P787 nanospheres exhibited a more

pronounced increase of Mw than PEC vectors. Volland

et al have observed that the initial molecular weight

of the irradiated polymer had an influence on the

cleavage mechanism. This phenomenon is called the

cage-effect of primary formed radicals. In fact, the

chain flexibility of PCL will decrease with increasing

molecular weight, leading to primary radical products

which will either recombine or undergo further

reactions depending on lifetime of the radicals formed.

Longer chains lengths of Tone P787 will thus promote

recombinations?.

The scission-cross-linking processing of PCL seems

to be pH dependent, as we observed a greater increase

of Mw in pH 7 buffered formulations, compared to

acidic samples. Surfactants also seem to play a role. In

fact, whatever the pH, y radiation induced a lower Mw

increase in nanospheres prepared with PF127 than

PF68. This could be due to the physico-chemical

properties of the surface active agent. PF68 is more

hydrophilic as can be seen from its HLB of 29

compared with 22 in the case of PF127. The penetration

of water in PF68 nanospheres and the subsequent

radical formation should thus be emphasized.

Furthermore, polyoxyethylene-polyoxypropylene

chains of PF68 are shorter than PF127. This may

promote the entanglement of polymer and surfactant

chains, thus leading to easier cross-linking. The weak

HLB of CRH 40 (= 15) compared with both synperonics

could also explain the lower increase of Mw in pH 7

CRH40 nanospheres.

Figure 3 represents pH evolution after y irradiation.

As opposed to autoclaving, the pH decrease was more

pronounced in non-buffered acidic nanospheres than

in buffered neutral formulations, This evolution was

almost the same for every polymer/surfactant

association, which means a 0.35 unit decrease in

buffered nanospheres and a 1.2 to 1.5 loss in acidic

preparations.

Table 3 summarizes pH evolution, observed after

irradiation of several formulations prepared with all

nanosphere constituents except the polymer. All

parameters tested remained unchanged, except a

decrease of pH of about 0.35 unit in buffered samples.

Thus, the acidification noted in pH 4 irradiated

nanospheres may result from the ester bond scission of

Biomaterials1997,Vol. 18 No. 4


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Sterilization of poly(e-caprolactone) nanospheres: V. Masson et a/.

60

pti4

pH4 HEC

pH7 buffer pH7 buffer HEC

Figure 2 Molecular weight (Mw) evolution of poly(E-caprolactone) inside nanospheres after y irradiation (u PEUPF68, a

PECIPF127, 0 PECICFiH40, one P787KXH40).

PH4

pH4 HEC

pH7 buffer

pH7 buffer HEC

Figure 3 pH evolution in irradiated nanospheres (m PEC/PF68,0 PEUPF127, 0 PECKRH40, one P787KRH40)

Table 3 pH evolution of irradiated samples, containing all nanosphere constituents except poly(E-caprolactone) (n = 3)

Surfactant

Sterilization

PF68

Non-

sterilized

3

irradiated

PF127

Non-

sterilized

-.

{rradiated

CRH40

Non-

sterilized

li

irradiated

PH 4 3.03 3.07 2.99 3.13 3.08 3.09

pH 7 buffer

7.08 6.75

7.08

6.73 7.07

6.70

the polymer with subsequent apparition of free

carboxylic endgroups. As no Mn value could be

calculated from chromatograms, we did not visualize

this phenomenon.

The lower decrease of pH in buffered neutral

nanospheres is likely to be a result of the better stability

of poly(caprolactone) in this condition, compared with

acidic medium. In fact, it is well known that polyesters

undergo acid-base catalysed hydrolysis. This may

converge with radical chain scission during irradiation.

It should be noted that pH decrease of pH 4

formulations was more pronounced after ; irradiation

compared with autoclaving. As no variation of Mw

Biomaterials 1997. Vol. 18 No. 4

was

noted after heat treatment, the acidification

observed after radiation sterilization could well be the

result of a larger poly(s-caprolactone) hydrolysis.

Furthermore, the decrease of pH does not seem to be

influenced by the surfactant used, as opposed to the

evolution of Mw. The surface active product probably

does not play any role in polymer chain scission,

whereas it acts directly in cross-linking.

In spite of the important modification of poly(a-

caprolactone) characteristics, no significant variation

of nanosphere diameter was observed. Anyway, some

authors have shown that radiation doses of 2 to 4 Mrad

were sufficient to convert the highly ductile PCL into a


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Sterilization of poly(e-caprolactone) nanospheres: V. Masson et al.

333

Table 4 Evolution of PEC nanospheres mean size and concentra0on before and after filtration (SD = standard deviation) (n = 3)

PF68

PF127

CRH40

Non- Filtered

Filtered

Non-

Filtered

Filtered Non-

Filtered

Filtered

Filtered

Zlrn

Y;rn

filtered

i;rn

Oil filtered

2pm

i;rn

2,m

+ 0.45 pm

+ 0.45pm

+ 0.45pm

+ 0.45pm + 0.45pm

+ 0.45pm

+ 0.2 pm + 0.2pm +0.2fim

Mean size 165k46

169*52 165548 169*48 168 3

165

k 47

133*29 137i36 128f29

fSD

T ) 65.04

65.50 65.69 65.90 66.60 66.89

68.49 69.12 69.24

f 0.40

& 0.28 f0.12 Zt 0.44 * 0.46 ZIZ .36

i 0.38 It 0.10 f 0.33

Remaining -

99.3 98.6 - 97.7 96.8

- 97.2 96.7

nanosphere

ZIZ .96 f 0.40 f1.54 Zt1.19

+Z0.38 i 1.27

cont. ( )

very brittle polymer.

This very early ductile/brittle

transition was interpreted as a result of cleavage of

constrained tie molecules, gradually untying the

crystalline domains and eliminating the load-bearing

elements53Z56. Thus, y radiation may influence the

stability of the vector or the release rate of associate

drugs34-36.

Sterile filtration

Table 4

presents mean diameters and turbidimetric

measurements obtained before and after filtration of

pH 4 PEC nanospheres.

Sterile filtration was easily carried out on all PEC

nanosphere samples. No important variation of particle

diameter or suspension transmission was observed.

Nevertheless, this method requires a prefiltration on

2pm and

0.45pm

filters and cannot be used for

viscous preparations. As opposed to autoclaving or

y irradiation, sterile filtration is the only process which

ensures conservation of the physicochemical character-

istics of the vector. Liposome?Go and microemul-

sions13B61 were thus sterile filtered in many reports. In

any case, no studies were undertaken on nanospheres,

as several stated that these systems were too similar in

size to contaminants and pore size of filters to allow

the use of this method34*36.

In this work, it is clearly shown that a sterile filtration

can be applied to polymeric nanospheres if the mean

diameter is sufficiently below 200nm, and if sample

viscosity is not too high.

ON LUSIONS

Any ophthalmic product must be free from harmful

microorganisms. For this purpose, terminal steriliza-

tion procedures are generally preferred over aseptic

processing. The more convenient and first tested

method is moist sterilization or autoclaving. This

process induced a massive aggregation of poly(s-

caprolactone)

nanospheres prepared with

cremophor RH40, and a yellowish colouration in

buffered samples, as a result of glucose oxidation.

A decrease of pH was observed in all preparations

and seems to be the result of an oxidation of

surfactant and, in some samples, of glucose. In any

case, no modification was noted on polymer

characteristics,

and the diameter of nanospheres

prepared

with Pluronic remained unchanged

throughout this experiment.

y radiation induced thimerosal degradation and HEC

depolymerization. Furthermore, a sharp increase of

Mw of poly(s-caprolactone) was observed in all

nanosphere preparations. This may result from a cross-

linking of polymer chains according to a radical

process induced by irradiation. No significant variation

was noted on nanosphere mean sizes.

Finally, sterile filtration was tested, as this process is

the only one which ensures the conservation of vector

characteristics. In fact, this method was applied with

success on PCL nanospheres with a diameter below

200nm. no filtration could be done on Tone P787

particles as their mean size was about 230nm.

Formulations containing HEC were equally too viscous

to allow this process.

Two methods could thus be proposed for the

sterilization of poly(s-caprolactone) nanospheres. The

first, sterile filtration, can be used if the nanosphere

diameter is small enough to diffuse through a 0.2 pm

filter. The second, moist sterilization, can be applied

to Pluronic nanospheres, containing tonicity agents

other than glucose. The modifications of polymer

properties induced after y irradiation are too great for

this to be used for nanosphere sterilization. In fact,

even if this evolution did not affect particle diameter,

it may alter their subsequent stability and drug release

characteristics.

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Biomaterials Volume 18 issue 4 1997 [doi 10.1016%2Fs0142-9612%2896%2900144-5] V. Masson; F. Maurin;...

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